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00c90f5ce440509eedb53d47542b880c3ebbee11798367aba2b352334bc8676b/preprint/images_list.json

@@ -0,0 +1,34 @@
+[
+    {
+        "type": "image",
+        "img_path": "images/Figure_1.jpg",
+        "caption": "Structure of P116 and its localization in Mycoplasma pneumoniae cells. a) Phase contrast (PhC) immunofluorescence microscopy images of M. pneumoniae cells using labeling with polyclonal antibodies against the ectodomains of adhesin P1 (top row; used as a reference) and P116 (bottom row). Labelling for P1 concentrates at the tip of the cell, while for P116 it covers the whole surface homogenously.\nb) Two views of the cryoEM density map of the complete extracellular region of the P116 dimer at 3.3 A\u030a resolution, 90 degrees apart. The homodimer is held together by the dimerization interface (shown in pink). The core domains have four contiguous antiparallel helices (shown in blue) and a \u03b2-sheet with five antiparallel strands (shown in orange). The N-terminal domain is shown in green. The top view displays a huge cavity that is fully accessible to solvent. The cleft providing access to the cavity spans the whole core domain. Each monomer also has a distinct protrusion (shown in blue as part of the antiparallel \u03b1-helices).",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_2.jpg",
+        "caption": "P116 structure and hydrophobic areas\na) Ribbon model of the P116 monomer, colored as in Fig. 1. The overall shape of the structure corresponds to a left hand, with the four antiparallel \u03b1-helices representing fingers (shown in blue), and the bridge helix and \u03b2-sheet of five antiparallel strands representing the palm. The N-terminal domain, which is very flexible, corresponds to the thumb. The dimerization helices (shown in pink) correspond to the wrist.\nb) The overall topology of P116. The N-terminal and core domains of P116 share a similar topology, which suggests that P116 might have been generated by duplication of an ancestor domain.\nc) The hydrophobic map of the P116 homodimer shows that the cavity in the core domain is hydrophobic.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_3.jpg",
+        "caption": "Purified P116 is filled with ligands and displays a large conformational variation compared to empty P116\na) Cross-section through the core domain of original P116 exposes a series of elongated densities (shown in red), which cannot be accounted for by the structure. These densities are ~4 \u00c5 wide and 10\u201319 \u00c5 long and are surrounded by highly conserved hydrophobic residues. The cross-section also reveals that the core domain can be accessed dorsally and distally. The side view of the core domain shows that the densities are aligned to the bridge helix and away from the fingers (shown in red). The individual fingers are indicated with digits 1 to 3 (finger 4 is not visible in this illustration).\nb) Overlay between empty and full P116. Side view of the cross-section surface view of the empty and full P116 shows that the fingers (in purple) have come closer to the core domain, massively reducing the available volume. Their new position is markedly different compared to the full P116 (shown in light blue). Finger 1 moved 8\u00c5 sideways and towards the core, finger 2 has moved 13\u00c5 towards the core and Finger 3 has moved 12\u00c5 towards the core. The volume in the empty P116 is not sufficient to accommodate ligands anymore.\nc) In the ribbon presentation the conformation differences between the empty and full P116 structures can be seen. All four fingers (antiparallel \u03b1-helices) have moved towards the inner part of the hand (individual distances are indicated filled conformation in light blue, empty conformation in purple).\nd) Two cryoEM classes reveal a wringing movement of P116. Comparison of the two density maps (superimposed with the ribbon diagram of the structure) shows that the wringing movement of P116 allows for the two hydrophobic cavities in the dimer to face almost opposite directions. The top view on the left shows both cavities facing in one direction, while the top view on the right shows the cavities rotated ~80 degrees to each other.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_4.jpg",
+        "caption": "Analysis of the lipid spectrum and uptake of P116\na) MALDI-TOF mass spectrum of original P116 sample (linear mode, high mass range), showing a dominant peak at the 105 kDa corresponding to the singly charged full protein, as well as the charges states two, three and four.\nb) Stacked MALDI-TOF mass spectra (reflector mode, low mass range) of the originally purified P116 (purple, back), the empty P116 (black, middle) and the refilled P116 sample (orange, front) showing a change in the lipid distribution among the samples. c) and d) Hierarchical clustering of lipid compounds identified in positive (c) and negative (d) ion mode lipidomics (LC-ESI-IMS-MS/MS) analyses, showing differential distributions of lipid compositions in original P116 (first column), emptied P116 (second column), refilled P116 (third column) and serum (fourth column), respectively. All data were normalized to the mTIC of all identified compounds in each sample and row-wise scaling was applied.\ne) When radiolabeled HDLs (here presented schematically) are incubated with P116, a net cholesterol transfer to P116 can be measured as indicated by the number at the flux arrow (for both free and esterified cholesterol).\nf) CryoEM analysis of empty P116 incubated with HDL shows that P116 binds HDLs between its N-terminal and core domains and is refilled. P116 is attached to HDL through its distal core access. Due to the flexibility of P116 and the variability of HDL, only one subunit of P116 can be seen at this threshold. Reducing the threshold causes the second subunit to appear.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    }
+]

00c90f5ce440509eedb53d47542b880c3ebbee11798367aba2b352334bc8676b/preprint/preprint.md

@@ -0,0 +1,247 @@
+# Abstract
+
+*Mycoplasma pneumoniae*, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically acquires essential lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are transported. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake.
+
+# Introduction
+
+*Mycoplasma pneumoniae* is a facultative intracellular human pathogen causing community-acquired pneumonia that can manifest severe systemic effects (1). Unlike other respiratory pathogens, *M. pneumoniae* has no approved vaccine (2). *Mycoplasmas* lack a cell wall and have the smallest known genomes (3). *M. pneumoniae*, with a 816 kb genome, is a model organism for a minimal cell (4). Many of the metabolic pathways required to synthesize essential products are absent, which makes an uptake by specialized mechanisms necessary. In fact, *M. pneumoniae* cannot synthesize several of the lipids that are important components of the cell membrane, such as sphingomyelin, phosphatidylcholine and cholesterol (5). Instead, it must take up lipids from the host environment and adapts its membrane composition depending on the medium in vitro (6–8). Cholesterol in particular, which is present in only a few prokaryotes, is essential for *M. pneumoniae* cells and several other *Mycoplasma spp.* (6). It is the most abundant lipid in the membranes, accounting for 35–50% of the total lipid fraction (6). To date, it is unclear how *Mycoplasma spp.* and other prokaryotic species achieve lipid uptake from the environment.
+
+In this work, we report the structural and functional characterization of P116, a strongly immunogenic and essential protein for the viability of *M. pneumoniae* cells. P116 was previously uncharacterized, although it has been reported to potentially contribute to adhesion to host cells (9). Despite the essential role of P116 the *M. pneumoniae* genome contains only a single copy of *p116* (*mpn213*). This is in contrast to the most immunogenic protein P1, which is not essential but contains multiple copies on the genome (10). To elucidate the role of P116, we first determined the structure of the ectodomain by single-particle cryo-electron microscopy (cryoEM). The structure has a novel fold (with no matches in the Protein Data Bank) featuring a uniquely large hydrophobic cavity that is fully accessible to solvent. Mass spectrometry and other analytical techniques identify ligands found in the cavity as several different lipids (incl. cholesterol), some of which are essential. Based on these findings, we describe the mechanism by which *Mycoplasmas* can extract lipids from the environment and possibly also deposit them in their own membrane, thus explaining the essential role of P116 for the survival of *M. pneumoniae* cells.
+
+# Results
+
+P116 is abundant on the cell surface  
+A construct predicted to span the whole ectodomain of P116 from *M. pneumoniae* (residues 30–957) was overexpressed in *Escherichia coli* and purified by His-tag affinity and gel filtration chromatography (Materials and Methods and *Supplementary Figure 1*). Immunolabeling with both polyclonal and monoclonal antibodies against this construct showed an intense and uniform distribution of labeling across the whole surface of *M. pneumoniae* cells (*Figure 1a*), with adhesion and motility unaffected by the antibodies (*Supplementary Table I and Supplementary Movie 1-3*). This distribution contrasts with that of P1, an adhesion protein that concentrates at the tip of the cell and has strong effects on adhesion and motility (*11, 12*).
+
+P116 has a novel fold with a lipid-accessible cavity  
+The structure of P116 (30–957) was determined by single-particle cryoEM at 3.3 Å resolution (according to the gold standard criterion of FSC 0.143; *Supplementary Table II*, *Supplementary Figure 2*). It is an elongated homodimer of ~240 Å along its longest axis, which adopts an arched shape with an arc-radius of 200 Å (*Figure 1b*, *Supplementary Movies 4, 5*). Each monomer consists of two distinct subunits: A N-terminal domain (residues 60–245), situated distal to the dimer axis, and a core domain (residues 246–867). Proximal to the dimer axis is the dimerization interface (*Figure 1b*, *Supplementary Figure 3*), which is very well resolved. In addition, the N-terminal domain has significant hinge mobility with respect to the core domain, which reduced the local resolution of the cryoEM map (*Supplementary Figure 2*), making model building difficult for the most distal parts of the construct (see Materials and Methods and *Supplementary Figure 4*). The homodimer displays significant flexibility with many vibrational modes, as classification illustrates (*Supplementary Figure 5*). Finally, some residues at the N- and C-termini of the construct (30–59 and 868–957, respectively) were not visible in the cryoEM maps. The flexibility of the homodimer involves a change in the curvature of approximately 100 Å, wringing along the axis perpendicular to the dimer axis by ~80 degrees, and bending up to 20 degrees (*Supplementary Figure 5, Supplementary Movie 6*).
+
+The core domain resembles a half-opened left hand, with four contiguous antiparallel α-helices corresponding to the four fingers and the N-terminal domain the thumb (*Figure 2a*). The helices corresponding to the wrist form the dimer interface, and a conserved tryptophan residue (Trp681) interacts tightly with the neighboring monomer. In the variant Trp681Ala, the rate of dimers to monomers is 1:4, compared to only dimers without the mutation (*Supplementary Figure 3b*). The palm of the hand includes a long and well defined central α-helix, the bridge helix (residues 268–304), and a rigid β-sheet of five antiparallel strands that extends to the N-terminal domain (*Figure 2b*). The hand appears in a half-opened state with a large elongated cleft across the whole core domain (*Figure 2c*). The inner part of the hand (i.e. the fingers and palm) forms a large cavity that measures 62 Å proximal to distal and 38 Å anterior to posterior with a volume of ~18,000 ų. The cavity is completely hydrophobic although fully accessible to the solvent (*Figure 2c*, *Supplementary Movie 7*). In addition, the core has two access points, one at the dorsal side and one at the distal side (*Figure 3a*). Using the DALI server, we found only very weak structural relationships between P116 and all other experimentally determined protein structures available in the Protein Data Bank, which shows that P116 has a new, unique fold.
+
+The N-terminal domain is compact and organized around a cluster of aromatic residues, at the center of which is the only tryptophan residue of the domain (Trp121). The N-terminal and core domains of P116 superimpose for 126 equivalent residues (68% of the N-terminal domain), suggesting that P116 might have been generated by duplication of an ancestor domain. The common secondary structural elements in the N-terminal and core domains consist of a β-sheet and the two helices preceding the sheet (*Figure 2b*). The core domain is much larger than the N-terminal domain mainly due to two insertions containing twelve and four helices, respectively.
+
+For the inner part of the P116 core domain, the cryoEM maps show prominent elongated densities (with a length of 10–19 Å and a width of 4 Å) that fill most of the hydrophobic areas (*Figure 3a*, *Supplementary Movies 8, 9*). These elongated densities, which are unaccounted for, cannot be explained by the protein residues missing in the model. Instead, the mass excess of ~13 kDa, consistently measured by multiple angle light scattering (MALS) and mass spectrometry for P116 in different preparations, could be explained by the presence of ligand molecules bound to P116 (*Figure 4a*). Initial mass spectrometry analysis of the same samples from which the structure of P116 was determined (see Materials and Methods) showed the presence of several lipid species, including phosphatidylcholine and sphingomyelin, which are essential for *M. pneumoniae*³, and of wax esters (*Figure 4b and Supplementary Figure 6*).
+
+P116 orthologues were found in at least eight other *Mycoplasma* spp. including *M. genitalium* and *M. gallisepticum*. The amino acids lining the hydrophobic cavity are largely conserved (either identical or with similar characteristics) (*Supplementary Figure 7a*). Modeling the orthologues of P116 with AlphaFold (*14*) results in all the models having a similar tertiary structure, in which a large core domain is flanked by a smaller N-terminal domain, but the relative position of the domains does not closely match the experimental structure (*Supplementary Figure 7b*).
+
+The conformation of empty P116 cannot accommodate lipid binding  
+To obtain ‘empty’ P116 that was free of any bound ligands, we treated the P116 samples with the detergent Triton-X 100 (see below and Materials and Methods). Mass spectrometry confirmed a massive reduction of lipids in the sample (*Figure 4b*). The structure of the empty P116 sample was solved by cryoEM at 4 Å resolution (*Supplementary Figure 8*). Its overall topology is almost identical to that of the original P116 sample, with the difference that the cavity is closed as a result of fingers 1, 2 and 3 being closer to the palm by 8, 13 and 12 Å, respectively, and finger 4 moving 11 Å sideways to retain the distal core access to the palm (*Figure 3b*, *Supplementary Movies 10*, *11*, and *Supplementary Figure 9*). These changes reduce the volume within the core domain from ~18,000 ų to ~6,300 ų. The unoccupied volume between the fingers and palm reduces to two pockets that are large enough for lipids to pass through but appear unoccupied in the cryoEM density. A comparison of the filled and empty P116 structures shows that the original densities that were unaccounted for create massive steric clashes in the closed configuration of the fingers, demonstrating that the cavity can no longer accommodate lipids (*Supplementary Movie 12*). In the empty P116, the dimerization interface is shifted towards the dorsal side of the molecule by 10 Å, resulting in a contraction that changes the arc radius of the dimer from 500 to 600 Å and shifts the N-terminal domain towards the dimerization interface.
+
+Refilled P116 is structurally identical to the purified sample  
+We next refilled the empty P116 samples by incubating them either with fetal bovine serum (FBS) or with high-density lipoproteins (HDL) and then re-purified them by affinity chromatography. Media containing FBS is a common growing broth for *M. pneumoniae* cultures, although lipoproteins, in particular HDL, are efficient substitutes for serum in mycoplasma culture media, likely because lipoproteins can provide the key lipids, in particular cholesterol, which is essential for mycoplasma cells (*15*). We solved the structure of the refilled P116 samples at 3.5 Å resolution using cryoEM. The structure of the refilled P116 is practically identical at 3.5 Å resolution to the structure of the original P116 sample, including densities at the palm of the hand that can be assigned to ligands. Mass spectrometry of the refilled samples shows the clear presence of lipids (*Figure 4b*). Classes of subunits of the dimer show a wringing of ~80 degrees (*Figure 3d*, *Supplementary Figure 5* and *Supplementary Movie 6*).
+
+P116 is conformationally flexible  
+In the original P116, empty P116 and refilled P116 samples, the structure appears predominantly as a homodimer. In all cases, the homodimer exhibits significant flexibility. Most prominently, the empty structure has a different arc radius than those of the original and refilled structures. In the original and refilled structures, a wringing motion is visible: each monomer is twisted in the opposite direction along the axis perpendicular to the dimer axis (*Figure 3d*, *Supplementary Movie 6* and *Supplementary Figure 5*). In all P116 structures, the N-terminal domain is the most flexible. Within the core domain, temperature factors are higher at the fingertips, indicating the movement of the antiparallel α-helices. When the fingers approach the palm, this results in a closing of the hand and a clash with the densities therein (*Supplementary Movie 12*).
+
+P116 ligands include essential lipids  
+We next set out to characterize the possible ligands within P116. We first measured the rate of radioactivity transfer to P116 after incubation with HDL particles containing either tritium-labeled cholesterol ([³H]cholesterol) or tritium-labeled cholesteryl oleate as a representative of cholesterol esters (*Table I*). A significant fraction of the HDL-[³H]-radiotracer was detected in the post-incubated and purified P116 fractions, indicating a net transfer of both cholesterol and cholesterol ester between HDL and P116. The total absence of the most abundant HDL protein (APOA1), cross-checked by immune detection, verified that no HDL remnants had contaminated the purified P116 fractions. The highest rate of radiotracer transfer was achieved when [³H]cholesterol-containing HDLs were mixed with empty P116. Transfer of [³H]cholesterol was also present, although reduced, when the original P116 was incubated with labeled HDL. Transfer of [³H]cholesterol esters to P116 would require a direct interaction between HDL and P116, as these esters are buried in the core of the HDL particles (*Table I*). Passive cholesterol transport has been reported from cellular membranes to HDL or from LDL to HDL (*16*), but the concept that bacteria can exploit such a mechanism is completely new. The net flux of cholesterol is bidirectional and is governed by the cholesterol gradient between acceptor and donor molecules.
+
+We then conducted a detailed liquid chromatography-coupled mass spectrometry (LC-MS) analysis. We identified more than 500 lipid species in the samples and found striking differences between the original, empty and refilled P116 samples (*Figure 4c, 4d*). Characterization of the lipids in the original and refilled P116 samples showed the presence of phosphatidylcholine and sphingomyelin lipids, among others, which are essential for *M. pneumoniae*. While these analyses found wax esters in the original P116, far fewer were found in the refilled P116. Wax esters are not known to be required by *M. pneumoniae*, although some pathogenic bacteria use wax esters as a carbon source (*17, 18*). However, wax esters are part of the cultivation medium of the *E. coli* strain in which P116 was produced. These findings are in agreement with the fact that *M. pneumoniae* takes up and incorporates many lipid species and adapts its membrane composition to the available lipid spectrum. In the P116 samples refilled from FBS, a clear accumulation of the essential lipids phosphatidylcholine and sphingomyelin, as well as cholesterol molecules, can be seen (*Figure 4c, 4d* and *Supplementary Table III*). These findings are in strong agreement with the functional data from the tritium-labeled cholesterol assay. Taken together, our lipidomics analyses revealed that P116 can bind to different lipid species. While in purified P116, PCs and SMs comprised only a small part of the present lipids, the most abundant species being PGs, PEs and wax esters, the refilled P116 preferentially bound to PCs, SMs, and cholesterols. Notably, the composition of the lipid species in the refilled P116 was strikingly different than the serum lipid distribution. For example, highly abundant uncharged TGs did not bind to P116. Thus, P116 although displaying a large bandwidth of lipid uptake, it does show a preference for selective lipid species (*Figure 4c, 4d* and *Supplementary Table III*).
+
+P116 binds HDL between its N-terminal and core domains  
+Next, we performed cryoEM on a sample containing empty P116 and HDL. Of ~46,000 particles that were identified as HDL, ~25,000 were attached to P116. The resulting density at a resolution of 9 Å shows P116 interacting directly with HDL at the region between the N-terminal domain and the core (*Figure 4f*). In the reconstruction the density of P116 resembles the filled conformation, and the structure can be well fitted to the density map. Cryo-electron tomograms of whole *M. pneumoniae* cells indicate a similar arrangement of P116 with respect to the *Mycoplasma* membrane, although an unambiguous identification of the involved complexes is challenging due to the modest resolution (*Supplementary Figure 10*).
+
+# Discussion
+
+P116 is essential for the viability of the human pathogen *M. pneumoniae* (4) and is the target of a strong antigenic response in infected patients (19). The P116 structure has a previously unseen fold with a uniquely large hydrophobic cavity filled with ligands. Mass spectrometry and radioactivity transfer experiments confirm a lipid extraction from serum (FBS) and HDL. Further, the ligands are identified as essential lipids for the survival of the cells. In fact, we found a high specificity towards Cholesterol, PCs and SMs, which represent the most abundant membrane lipids in *M. pneumoniae* (8). Crosslinking mass spectrometry studies indicate one weak aminoacid-pair interaction between P116 and MPN161 (a protein of unknown function) (20). Thus, while the involvement of other proteins in incorporating the extracted lipids into the *Mycoplasma* membrane cannot be excluded, it appears likely, given the observed conformational cycle upon lipid uptake, that P116 is also responsible for incorporation. Altogether, the P116 structure, along with our insights into different P116 conformations and the P116 complex formation with HDL, reveals a mechanism by which *Mycoplasmas* extract lipids from their environment and most likely incorporate them into their own membrane.
+
+The transition from a full to an empty P116 molecule involves a ~ 70% volume reduction of the hydrophobic cavity in concert with a wringing motion of the core domains. During this wringing motion, in which the monomers are each twisted in the opposite direction around their long axis, the hydrophobic cavities face almost opposite directions. Since, the N-terminal domain is in close proximity to the C-terminus with which the protein is anchored in the *Mycoplasma* membrane in vivo, the core domain is the one experiencing the high flexibility seen in our data sets. This enables an alternating motion of the core domain, in which each time one monomer of the core domain faces the *Mycoplasma* membrane (i.e. the one transferring lipids to the membrane) and the other monomer faces the environment (i.e. the one extracting lipids from the environment). This wringing motion can be repeated in a continuous manner. In this way, P116 could undergo a rolling movement on the *Mycoplasma* membrane, thus facilitating the transport of cholesterol and other essential lipids in an apparently simple and newly discovered way for lipid transporters.
+
+*Mycoplasmas* have a minimal genome and are capable of incorporating many different lipids into their membrane (6, 7). The lipid-binding versatility shown by P116 enables a single molecular system to cope with the transport of diverse lipids required by *Mycoplasmas*. Although only *Mycoplasmas* share genes with similar sequences to P116, other microorganisms that require uptake of lipids from the environment, including clinically relevant bacterial species such as *Borrelia burgdorferi* may have similar -not yet discovered- systems to regulate their cholesterol homeostasis. Whether P116 shares functional similarities with other transfer proteins such as human cholesteryl ester transfer and phospholipid transfer proteins (21, 22) requires further investigation. However, the diversity and amount of lipids that P116 can bind appear to be unmatched by any other known prokaryotic or eukaryotic lipid carrier. Interestingly, despite the broad lipid range P116 still shows a high specificity, largely enriching certain lipids (SM, PC and cholesterol) while excluding others (TGs). This new understanding of bacterial lipid uptake opens possibilities for treatment of mycoplasma infections and may, for the first time (2), allow the creation of a vaccine against *Mycoplasma pneumoniae*.
+
+# References
+
+1. S. Tsiodras, I. Kelesidis, T. Kelesidis, E. Stamboulis, H. Giamarellou, Central nervous system manifestations of Mycoplasma pneumoniae infections. The Journal of infection. **51**, 343–354 (2005), doi: 10.1016/j.jinf.2005.07.005.
+
+2. Z. Jiang, S. Li, C. Zhu, R. Zhou, P. H. M. Leung, Mycoplasma pneumoniae Infections: Pathogenesis and Vaccine Development. Pathogens (Basel, Switzerland). **10** (2021), doi: 10.3390/pathogens10020119.
+
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+
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+
+5. E. Gaspari *et al.*, Model-driven design allows growth of Mycoplasma pneumoniae on serum-free media. NPJ systems biology and applications. **6**, 33 (2020), doi: 10.1038/s41540-020-00153-7.
+
+6. S. Razin, M. Argaman, J. Avigan, Chemical composition of mycoplasma cells and membranes. Journal of general microbiology. **33**, 477–487 (1963), doi: 10.1099/00221287-33-3-477.
+
+7. O. Leon, C. Panos, Long-chain fatty acid perturbations in Mycoplasma pneumoniae. Journal of bacteriology. **146**, 1124–1134 (1981), doi: 10.1128/jb.146.3.1124-1134.1981.
+
+8. R. Bittman, Mycoplasma membrane lipids. Chemical composition and transbilayer distribution. Sub-cellular biochemistry. **20**, 29–52 (1993), doi: 10.1007/978-1-4615-2924-8_2.
+
+9. H. F. Svenstrup, P. K. Nielsen, M. Drasbek, S. Birkelund, G. Christiansen, Adhesion and inhibition assay of Mycoplasma genitalium and M. pneumoniae by immunofluorescence microscopy. Journal of medical microbiology. **51**, 361–373 (2002), doi: 10.1099/0022-1317-51-5-361.
+
+10. C. L. McGowin, P. A. Totten, The Unique Microbiology and Molecular Pathogenesis of Mycoplasma genitalium. The Journal of infectious diseases. **216**, S382-S388 (2017), doi: 10.1093/infdis/jix172.
+
+11. D. C. Krause, D. K. Leith, R. M. Wilson, J. B. Baseman, Identification of Mycoplasma pneumoniae proteins associated with hemadsorption and virulence. Infection and immunity. **35**, 809–817 (1982), doi: 10.1128/iai.35.3.809-817.1982.
+
+12. J. B. Baseman, R. M. Cole, D. C. Krause, D. K. Leith, Molecular basis for cytadsorption of Mycoplasma pneumoniae. Journal of bacteriology. **151**, 1514–1522 (1982), doi: 10.1128/jb.151.3.1514-1522.1982.
+
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+20. F. J. O'Reilly *et al.*, In-cell architecture of an actively transcribing-translating expressome. *Science (New York, N.Y.)*. **369**, 554–557 (2020), doi: 10.1126/science.abb3758.
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+21. L. Zhang *et al.*, Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein. Nature chemical biology. **8**, 342–349 (2012), doi: 10.1038/nchembio.796.
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+22. M. Zhang *et al.*, Structural basis of the lipid transfer mechanism of phospholipid transfer protein (PLTP). *Biochimica et biophysica acta. Molecular and cell biology of lipids*. **1863**, 1082–1094 (2018), doi: 10.1016/j.bbalip.2018.06.001.
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+23. G. Köhler, C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. 1975. *Journal of immunology (Baltimore, Md.: 1950)*. **174**, 2453–2455 (2005).
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+35. S. Seto, T. Kenri, T. Tomiyama, M. Miyata, Involvement of P1 adhesin in gliding motility of Mycoplasma pneumoniae as revealed by the inhibitory effects of antibody under optimized gliding conditions. Journal of bacteriology. **187**, 1875–1877 (2005), doi: 10.1128/JB.187.5.1875-1877.2005.
+
+# Table I: Relative transfer of (esterified) cholesterol from HDL to P116
+
+| | % of [³H]cholesterol transferred/mL | nmol cholesterol transferred/mL/h | nmol cholesterol transferred/mg P116\* |
+|--- | --- | --- | ---|
+| HDL to empty P116 |  |  |  |
+| Free cholesterol | 13.12 | 13.52 | 59.49 (6.3) |
+| Esterified cholesterol | 6.98 | 7.22 | 31.75 (3.3) |
+| HDL to original P116 |  |  |  |
+| Free cholesterol | 7.89 | 7.42 | 32.63 (3.4) |
+| Esterified cholesterol | 6.32 | 6.01 | 26.44 (2.8) |
+
+* Numbers in parentheses are the estimated number of cholesterol molecules transferred per P116 subunit (assuming a Mw of ~105 KDa for the construct).
+
+# materials and methods
+
+## Bacterial strains, tissue cultures and growth conditions
+
+*M. pneumoniae* M129 strain was grown in cell culture flasks containing SP4 medium and incubated at 37°C and 5% CO₂. Surface-attached mycoplasmas were harvested using a cell scraper and resuspended in SP4 medium. To grow mycoplasma cells on IBIDI 8-well chamber slides, each well was seeded with about 10⁵ CFUs and incubated for 12–24 h in 200 μL SP4 supplemented with 3% gelatin.
+
+NSI myeloma cells (23) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 50 μg mL⁻¹ gentamycin (complete RPMI). Hybridomas were selected in complete RPMI supplemented with HAT media and BM-Condimed (Sigma Aldrich, St. Louis, USA).
+
+## Cloning, expression, and purification of P116 constructs
+
+Regions corresponding to the MPN213 gene from *M. pneumoniae* were amplified from synthetic clones using different primers for each construct: P116F₃₀ and P116R₉₅₇ for P116(30–957); P116F₁₃ and P116R₉₅₇ for P116(13–957); P116F₂₁₂ and P116R₈₆₂ for P116(212–862); and P116W₆₈₁ to introduce mutation W681A. PCR fragments were cloned into the expression vector pOPINE (gift from Ray Owens; plasmid #26043, Addgene, Watertown, USA) to generate constructs, with a C-terminal His-tag. Recombinant proteins were obtained after expression at 22°C in B834 (DE3) cells (Merck, Darmstadt, Germany), upon induction with 0.6 mM IPTG at 0.8 OD₆₀₀. Cells were harvested and lysed by French press in binding buffer (20 mM TRIS-HCl pH: 7.4, 40 mM imidazole and 150 mM NaCl) and centrifuged at 49,000 × g at 4°C. Supernatant was loaded onto a HisTrap 5 ml column (GE Healthcare, Chicago, USA) that was pre-equilibrated in binding buffer and elution buffer (20 mM TRIS-HCl pH: 7.4, 400 mM imidazole and 150 mM NaCl). Soluble aliquots were pooled and loaded onto a Superdex 200 GL 10/300 column (GE Healthcare, Chicago, USA) in a protein buffer (20 mM TRIS-HCl pH 7.4 and 150 mM NaCl).
+
+To obtain empty P116, 2.6% Triton X-100 was added to the protein sample and incubated for 1.5 h at room temperature. Subsequent purification followed the same methodology described above, but also included a wash step with the binding buffer plus 1.3% Triton X-100, followed by extensive washing with at least 20 column volumes of wash buffer (20 mM TRIS-HCl pH: 7.4, 20 mM imidazole) before eluting the samples from the column. P116 was concentrated with Vivaspin 500 centrifugal concentrators (10,000 MWCO PES, Sartorius, Göttingen, Germany) to a final concentration of >0.5 mg/mL.
+
+To refill P116 with lipids, the empty protein was incubated with approximately 1 ml FBS per mg P116 for 2 h at 30°C while still bound on the column. After extensive washing with at least 40 column volumes of wash buffer, elution and concentration were performed as described above.
+
+## HDL isolation and determination of cholesterol transfer rate
+
+Human HDL (density 1.063–1.210 g/mL) was isolated from plasma of healthy donors via sequential gradient density ultracentrifugation, using potassium bromide for density adjustment, at 100,000 g for 24 h with an analytical fixed-angle rotor (50.3, Beckman Coulter, Fullerton, CA, USA). The amount of cholesterol and apolipoprotein A1 were determined enzymatically and by an immunoturbidimetric assay, respectively, using commercial kits adapted for a COBAS 6000 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland). Radiolabeled HDLs were prepared as previously described (24). Briefly, 10 μCi of either [1,2-³H(N)] free cholesterol or [1,2-³H(N)]cholesteryl oleate (Perkin Elmer, Boston, MA) were mixed with absolute ethanol, and the solvent was dried under a stream of N₂. HDL (0.5 mL, 2.25 g/L of ApoA1) was added to the tubes containing the radiotracers, as appropriate, and then incubated for 16 h in a 37°C bath. The labeled HDLs (both ³H-cholesterol-containing and ³H-cholesteryl oleate-containing HDLs) were re-isolated by gradient density ultracentrifugation at 1.063–1.210 g/mL and dialyzed against PBS via gel filtration chromatography. Specific activities of ³H-cholesterol-containing and ³H-cholesteryl oleate-containing HDLs were 1221 and 185 counts per minute (cpm)/nmol, respectively. The cholesterol transfer to P116 (1 g/L) was measured after adding either [³H] free cholesterol-containing or [³H]cholesteryl oleate-containing HDL (0.5 g/L of APOA1) and incubating for 2 h at 37°C. HDL and P116 were separated by a HisTrap HP affinity column. The radioactivity associated with each P116 and HDL fraction was measured via liquid scintillation counting. The percentage of [³H]cholesterol transferred per mL was determined for each condition. The specific activities for each radiotracer were used to calculate the amount of free cholesterol and cholesteryl ester transferred from HDL to P116.
+
+## Size exclusion chromatography and multi-angle light scattering (SEC-MALS)
+
+Molecular weights were measured from P116 samples using a Superose 6 10/300 GL (GE Healthcare, Chicago, USA) column in a Prominence liquid chromatography system (Shimadzu, Kyoto, Japan) connected to a DAWN HELEOS II multi-angle light scattering (MALS) detector and an Optilab T-REX refractive index (dRI) detector (Wyatt Technology, Santa Barbara, USA). ASTRA 7 software (Wyatt Technology) was used for data processing and analysis. An increment of the specific refractive index in relation to concentration changes (dn/dc) of 0.185 mL/g (typical of proteins) was assumed for calculations.
+
+## Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF)
+
+All samples were mixed in a 1:1 ratio with either DHB or sDHB (Bruker Daltonics, Germany) matrix solution (50 mg·ml⁻¹ in 50% Acetonitrile (ACN), 50% water and 0.1% TFA). Subsequently 1 μl aliquots of the mixture were deposited on a BigAnchor MALDI target (Bruker Daltonics, Germany) and allowed to dry and crystallize at ambient conditions. Unless stated otherwise, all reagents and solvents were obtained from Sigma Aldrich, Germany.
+
+MS spectra were acquired on a rapifleX MALDI-TOF/TOF (Bruker Daltonics, Germany) in the mass range from 20.000–120.000 m/z in linear positive mode and in the mass range from 100–1600 m/z in reflector positive mode. The Compass 2.0 (Bruker, Germany) software suite was used for spectra acquisition and processing.
+
+## Lipidomics analysis (LC-TIMS-MS/MS)
+
+Samples were extracted using a modified MTBE/Methanol extraction protocol, and submitted to LC-nanoESI-IMS-MS/MS analysis using a Bruker NanoElute UHPLC coupled to a Bruker TimsTOF Pro 2 mass spectrometer operated in DDA-PASEF mode. In brief, 40 min gradients on PepSep C18 columns (1.9A, 75µm ID, 15cm length) were recorded in positive and negative ion mode. Data were analysed using the MS-DIAL pipeline (version 4.9).
+
+## Single-particle cryoEM
+
+For single-particle cryoEM, a 3.5 µl drop of purified P116 (100–400 µg/mL in 20 mM Tris, pH 7.4 buffer or 600 µg/mL in 20 mM Tris, 2 mM CHAPSO, pH 7.4 buffer) or P116 mixed with HDL (250 µg/mL P116 and 1116 µg/mL HDL in 20 mM Tris, pH 7.4 buffer) was applied to a (45 s) glow-discharged R1.2/1.3 C-flat grid (Electron Microscopy Science, Hatfield, USA), and plunge-frozen in liquid ethane (Vitrobot Mark IV, Thermo Scientific, Waltham, USA) at 100% relative humidity, 4 °C, nominal blot force –3, wait time 45 s, with a blotting time of 12 s. Before freezing, Whatman 595 filter papers were incubated for 1 h in the Vitrobot chamber at 100% relative humidity and 4°C.
+
+Dose-fractionated Movies of P116, P116 refilled and P116 mixed with HDL were collected with SerialEM v3.8 (25) at a nominal magnification of 130,000x (1.05 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios (Thermo Scientific, Waltham, USA) electron microscope equipped with a GIF Quantum S.E. post-column energy filter in zero loss peak mode and a K2 Summit detector (Gatan Inc., Pleasanton, USA). For P116, P116 refilled and P116 with HDL a total of 4376, 4019 and 3114 micrographs with 34, 29 and 30 frames per micrograph and a frame time of 0.2 s were collected. The camera was operated in dose-fractionation counting mode with a dose rate of ~8 electrons per Ų s⁻¹, resulting in a total dose of 50 electrons per Ų s⁻¹. Defocus values ranged from –1 to –3.5 µm.
+
+For P116 empty, dose-fractionated Movies were collected using EPU 2.12 (Thermo Scientific, Waltham, USA) at a nominal magnification of 105,000x (0.831 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios G2 electron microscope (Thermo Scientific, Waltham, USA), equipped with a BioQuantum-K3 imaging filter (Gatan Inc., Pleasanton, USA), operated in zero loss peak mode with 20 eV energy slit width. In total 15,299 micrographs with 50 frames per micrograph and frame time of 0.052 s were collected. The K3 camera was operated in counting mode with a dose rate of ~ 16 electrons per A² s⁻¹, resulting in a total dose of 50 electrons per Ų s⁻¹. Defocus values ranged from -0.8 to -3.5 µm.
+
+CryoSPARC v3.2 (26) was used to process the cryoEM data, unless stated otherwise. Beam-induced motion correction and CTF estimation were performed using CryoSPARC’s own implementation. Particles were initially clicked with the Blob picker using a particle diameter of 200–300 Å. Particles were then subjected to unsupervised 2D classification. For the final processing, the generated 2D averages were taken as templates for the automated particle picking, for the processing of P116 with HDL no template picking was performed. In total, 3,463,490, 4,532,601 particles, 2,930,863 particles and 262,981 particles were picked and extracted with a binned box size of 256 pixels for P116, P116 empty, P116 refilled and P116 with HDL respectively. False-positive picks were removed by two rounds of unsupervised 2D classification. The remaining 1,324,330 particles (P116), 1,140,275 particles (P116 empty), 1,311,526 particles (P116 refilled) and 46,277 particles (P116 with HDL) were used to generate an ab initio reconstruction with three classes followed by a subsequent heterogeneous refinement with three classes. For the final processing, 1,315,362 particles (P116), 633,332 particles (P116 empty), 1,311,526 particles (P116 refilled) and 46,277 particles (P116 with HDL) were used. For the remaining particles, the beam-induced specimen movement was corrected locally.
+
+The CTF was refined per group on the fly within the non-uniform refinement. The obtained global resolution of the homodimer was 3.3 Å (P116), 4 Å (P116 empty), and 3.5 Å (P116 refilled) (Supplementary Figure 2 & 8 and Supplementary Table II). To analyze the sample in regard to its flexibility the particles were subjected to the 3D variability analysis of cryoSPARC which was used to display the continuous movements of the protein.
+
+## Cryo-electron tomography of *M. pneumoniae*
+
+*M. pneumoniae* M129 cells of an adherently growing culture were scraped in a final volume of 1 ml of SP4 medium and washed three times in PBS. This solution was mixed with fiducial markers (Protein A conjugated to 5 nm colloidal gold: Cell biology department, University Medical Center Utrecht, The Netherlands). From this stock a 3.5 µl drop was applied to a (45 s) glow-discharged R1.2/1.3 C-flat grid (Electron Microscopy Science, Hatfield, USA), and plunge-frozen in liquid ethane (Vitrobot Mark IV, Thermo Scientific, Waltham, USA) at 100% relative humidity, 4 °C, nominal blot force –1, with a blotting time of 10 s.
+
+Tilt-series were recorded using SerialEM v3.8 (25) at a nominal magnification of 105,000x (1.3 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios (Thermo Scientific, Waltham, USA) electron microscope equipped with a GIF Quantum S.E. post-column energy filter in zero loss peak mode and a K2 Summit detector (Gatan Inc., Pleasanton, USA). The total dose per tomogram was 120 e⁻/ Ų, the tilt series covered an angular range from -60° to 60° with an angular increment of 3° and a defocus set at -3 µm. Tomograms were reconstructed by super-sampling SART (27) with a 3D CTF correction (28).
+
+## P116 model building and refinement
+
+The initial tracing of the core domain was performed manually with Coot (29). It contained numerous gaps and ambiguities that were slowly polished by alternating cycles of refinement using the “Real Space” protocol in the program Phenix (30, 31) and manual reinterpretation and rebuilding with Coot. The tracing and assignment of specific residues in the N-terminal domain were very difficult due to the low local resolution of the map for this domain, and only a partial interpretation was achieved. Using Robetta and AlphaFold (14) we obtained different predictions of the N-terminal domain structure using different parts of the sequence. The highest ranked predictions, selected using the partial experimental structure already available, were obtained with AlphaFold for residues 81–245, which allowed us to complete the building of the N-terminal domain according to the cryoEM map. The RMS deviation between the AlphaFold prediction and the experimental model was 2.6 Å for 104 (63%) structurally equivalent residues. Some residues at the N-end of the N-terminal domain were difficult to identify and were represented as alanines in the final model. The whole P116 model was then refined using Phenix, and the final refined structure was deposited in the EMDB with code XXXX (Supplementary Table II).
+
+## Polyclonal and monoclonal antibody generation
+
+Two BALB/C mice were serially immunized with four intraperitoneal injections, each one containing 150 μg of recombinant P116 ectodomain (residues 30–957) in 200 μL of PBS with no adjuvants. The last injection was delivered four days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells (23) to obtain stable hybridoma cell lines producing monoclonal antibodies, as previously described (32). Supernatants from hybridoma cell lines derived from single fused cells were first investigated by indirect ELISA screening against the recombinant P116 ectodomain. Positive clones were also tested by Western blot against protein profiles from *M. pneumoniae* cell lysates and by immunofluorescence using whole, non-permeabilized *M. pneumoniae* cells (see below). Only those clones with supernatants revealing a single 116 kDa band in protein profiles and also exhibiting a consistent fluorescent staining of *M. pneumoniae* cells were selected and used in this work. Polyclonal sera were obtained by cardiac puncture of properly euthanized mice just before splenectomy and titred using serial dilutions of the antigen. The titer of each polyclonal serum was determined as the IC₅₀ value from four parameter logistic plots and found to be approximately 1/4000 for both sera. Polyclonal anti-P1 antibodies were obtained by immunizing two BALB/C mice with recombinant P1 proteins (33), respectively, as described above. The titers obtained for polyclonal anti-P1 antibodies were approximately 1/2500 and 1/3000, respectively.
+
+## Immunofluorescence microscopy
+
+The immunofluorescence staining of mycoplasma cells on chamber slides was similar to previously described (34), with several modifications. Cells were washed with PBS containing 0.02% Tween 20 (PBS-T) prewarmed at 37°C, and each well was fixed with 200 μL of 3% paraformaldehyde (wt/vol) and 0.1% glutaraldehyde. Cells were washed three times with PBS-T, and slides were immediately treated with 3% BSA in PBS-T (blocking solution) for 30 min. The blocking solution was removed, and each well was incubated for 1 h with 100 μL of the primary antibodies diluted in blocking solution. For P116 polyclonal sera, we used a 1/2000 dilution; a 1/10 dilution was used for monoclonal antibodies from hybridoma supernatants. Wells were washed three times with PBS-T and incubated for 1 h with a 1/2000 dilution of a goat anti-mouse Alexa 555 secondary antibody (Invitrogen, Waltham, USA) in blocking solution. Wells were then washed three times with PBS-T and incubated for 20 min with 100 μL of a solution of Hoechst 33342 10 μg/μL in PBS-T. Wells were finally washed once with PBS-T and replenished with 100 μL of PBS before microscopic examination. Cells were observed by phase contrast and epifluorescence in an Eclipse TE 2000-E inverted microscope (Nikon, Tokyo, Japan). Phase contrast images, 4',6-diamidino-2-phenylindole (DAPI, excitation 387/11 nm, emission 447/60 nm) and Texas Red (excitation 560/20 nm, emission 593/40 nm) epifluorescence images were captured with an Orca Fusion camera (Hamamatsu, Hamamatsu, Japan) controlled by NIS-Elements BR software (Nikon, Tokyo, Japan).
+
+## Time-lapse microcinematography
+
+The effect of anti-P116 antibodies and anti-P1 polyclonal serum on mycoplasma cell adhesion was investigated by time-lapse cinematography of *M. pneumoniae* cells growing on IBIDI 8-well chamber slides. Before observation, medium was replaced with PBS containing 10% FBS and 3% gelatin prewarmed at 37°C. A similar medium has been used to test the effect of P1 antibodies on mycoplasma adhesion and gliding motility(35). After incubation for 10 min at 37°C and 5% CO₂, the slide was placed in a Nikon Eclipse TE 2000-E inverted microscope equipped with a Microscope Cage Incubation System (Okolab, Pozzuoli, Italy) at 37°C. Images were captured at 0.5 s intervals for a total observation time of 10 min. After the first 60 s of observation, the different antibodies were dispensed directly into the wells. The frequencies of motile cells and detached cells before the addition of antibodies were calculated from the images collected between 0 and 60 s of observation. The frequencies of motile cells and detached cells after the addition of antibodies were calculated from the images collected in the last minute of observation.
+
+# Supplementary Files
+
+- [sprankelEtAlSupplementaryNM.docx](https://assets-eu.researchsquare.com/files/rs-1814661/v1/e13dd6a8745645c57a47932c.docx)  
+  supplemental material
+
+- [SupplementarytableIII.xlsx](https://assets-eu.researchsquare.com/files/rs-1814661/v1/bf7240b7bd3b08b4dc118f34.xlsx)  
+  Supplementary Table III: Identified lipid compounds from of P116, P116-empty, P116-refilled and serum in positive and negative mode as used for heatmap generation.
+
+- [movie1AntiP1Mpn2.mov](https://assets-eu.researchsquare.com/files/rs-1814661/v1/106613edfbca919fac358e18.mov)  
+  movie 1
+
+- [movie2AntiP116Mpn2.mov](https://assets-eu.researchsquare.com/files/rs-1814661/v1/b5d846cf7bdde0944c143acc.mov)  
+  movie 2
+
+- [movie3PBScontrolMpn.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/4c19ab2a24e56f707afb7a02.mp4)  
+  movie 3
+
+- [movie4cryoEMP116dimer.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/a14f0d71cd5870a90a9ff0ae.mp4)  
+  movie 4
+
+- [movie5ribbonModelP116dimer.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/73e46fde9721ce5f5b449616.mp4)  
+  movie 5
+
+- [movie6P116wringing.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/3e866d0059d2d1c11ace6fcc.mp4)  
+  movie 6
+
+- [movie7P116hydrophobicityMap.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/e630efb25aee3ea8265097d4.mp4)  
+  movie 7
+
+- [movie8P116CrossSectionLigands.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/c0efa76a7716da7d2e056df4.mp4)  
+  movie 8
+
+- [movie9P116RiboonWithligands.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/66a8f8da4c0a1b58c1a9ed96.mp4)  
+  movie 9
+
+- [movie10P1116FullToEmptyContraction.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/cbef4a980cf01d37e0bb07ca.mp4)  
+  movie 10
+
+- [movie11P116FullToEmptyContractionDistalView.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/1870989dcb381526985732f7.mp4)  
+  movie 11
+
+- [movie12P116RibbonFullToEmptyContractionClasehs.mp4](https://assets-eu.researchsquare.com/files/rs-1814661/v1/0a7703e091e643f0c51b91ba.mp4)  
+  movie 12

059a54d253ab5af79d48ff1a3f02886f0511ffb2444cb09d7b8ed598ea46c8cb/preprint/images_list.json

@@ -0,0 +1,50 @@
+[
+    {
+        "type": "image",
+        "img_path": "images/Figure_1.jpeg",
+        "caption": "Interaction mapping.\na, Domain schemes ASCC3 and ASC1, domain borders and borders of fragments employed. Numbers on top, residues bordering domains/regions. Numbers on the right, ASCC3HRor ASC1 C-terminal residues. Numbers on the bottom (ASC1), borders of fragments used for interaction studies. NTR, N-terminal region; CR, NTR-NC connecting region; NC/CC, N-terminal/C-terminal cassettes; L, NC-CC linker; HR, helicase region; ZnF, zinc finger domain; LP, lasso peptide; ASCH, ASC1-homology domain.\nb-e, SDS-PAGE analyses of analytical SEC elution fractions monitoring the interaction of ASCC3HR with different regions of ASC1. Throughout all panels, equivalent elution fractions are vertically aligned. Molecular mass markers in kDa are shown on the left; protein bands are identified on the right. Coomassie stain, black outlines; silver stain, golden outlines. For stably interacting fragments, analytical SEC runs of the individual proteins are shown for comparison. For some analytical SEC runs, separate regions of the same gel were spliced together for display purposes (see Source Data file for uncropped gels). Dashed lines, splice lines.\nb, ASC1 stably binds ASCC3HR, but to ASCC3NTR.\nc, ASC11-80 does not stably bind ASCC3HR.\nd-e, All fragments containing the ZnF domain (ASC1152-581, ASC11-230, ASC1152-230) stably bind ASCC3HR.\nf, C-terminal fragments of ASC1 lacking the ZnF (ASC1281-403, ASC1403-581, ASC1281-581) do not stably bind ASCC3HR.\nExperiments were repeated independently at least three times with similar results.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_2.jpeg",
+        "caption": "CryoEM structure of a ASCC3HR-ASC1 complex.\na, Overview of the cryoEM reconstruction of the ASCC3HR-ASC1 complex colored by ASCC3HR cassettes and ASC1 domains/regions. If not mentioned otherwise, coloring in this and the following figures: NC, slate blue; CC, light gray; NC-CC linker, cyan; ZnF domain, brown; lasso peptide, orange; ASCH domain, gold-yellow. In this and the following figures: rotation symbols, orientation relative to Fig. 2a, left.\nb, Combined surface (ASCC3HR) and cartoon (ASC1) plot of the ASCC3HR-ASC1 complex model. Zn2+ ions, green spheres.\nc, Domain scheme (top) and orthogonal cartoon representation (bottom) of the ASCC3HR subunit of the ASCC3HR-ASC1 complex, colored by domains/regions (identical domain/region colors in NC and CC). Numbers on top, residues bordering domains/regions. Numbers on the left/right, ASCC3HR N/C-terminal residues. CR, NTR-NC connecting region, violet; RecA1, light gray; RecA2, dark gray; WH, black; HB, slate blue; HLH, red; IG, lime green; L, NC-CC linker, cyan.\nd, Close-up views of the interfaces of the ZnF domain (left), lasso-like peptide (middle) and ASCH domain (right) with ASCC3HR. Interacting residues are shown as sticks, colored by atom type, and labeled. In this and the following figures: Carbon, as the respective protein region; nitrogen, blue; oxygen, red. Dashed black lines, hydrogen bonds or salt bridges.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_3.jpeg",
+        "caption": "Interactions of ASC1 variants in cells.\na, Immuno-fluorescence microscopy of Flp-In\u00e4 T-REx\u00e4 293 cells stably expressing Flag-tagged ASC1 variants (identified on the top; N-terminal Flag-tag, left; C-terminal Flag-tag, right) after staining with \u03b1-Flag antibody (top rows) and DAPI (bottom rows), revealing nuclear and cytosolic localization of all ASC1 constructs. Scale bars, 10 \u00b5m.\nb, Western blots (WB) monitoring immuno-precipitation (IP) of ASCC1, ASCC2 and ASCC3 by the indicated N-terminally (left) or C-terminally (right) Flag-tagged ASC1 variants from the cell extracts.\nc, Western blots (WB) monitoring immuno-precipitation (IP) of ASCC3 by the indicated HA-tagged ASC1 variants (negative control, GFP). Wt, ASC1 wild type; \u0394ZnF, ASC1\u0394168-219; LLI-AAA, ASC1L174A-L180A-I190A; CC-AA, ASC1C171A-C184A.\nExperiments were repeated independently three times with similar results.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_4.jpeg",
+        "caption": "Activation of ASCC3HR helicase by ASC1.\na, Experimental setup for multiple-round stopped-flow/fluorescence-based unwinding assays. Gray sphere, helicase; star symbol, fluorophore (Alexa 488); red sphere, quencher (Atto 540 Q).\nb, Stopped-flow/fluorescence-based DNA unwinding assays, showing that ASC1, but not ASC11-230 or ASC1403-581, stimulates ASCC3HRhelicase activity. Data for ASCC3HR,D611A-based unwinding had been reported previously20 and are reproduced here to facilitate direct comparison.\nc, Multiple sequence alignments of conserved NTPase/helicase motifs (identified by letters or Roman numerals above the alignment) in human ASCC3, human SNRNP200 and yeast Slh1p (ASCC3 ortholog) N-terminal and C-terminal cassettes. Motifs involved in ATP binding, light gray; motifs involved in nucleic acid binding, gray; motifs involved in coupling of ATP and nucleic acid transactions, dark gray. Conserved motif II aspartate residues of ASCC3, which were altered to alanine to inactivate the NC or CC, magenta.\nd, As b, monitoring unwinding by ASCC3HR constructs, in which either the NC (D611A) or the CC (D1463A) are inactivated, alone or in the presence of ASC1, showing that both NC and CC exhibit helicase activities that are stimulated by ASC1.\ne, Apparent DNA-stimulated ATPase rates of ASCC3 constructs alone or in complex with ASC1 (indicated at the bottom). HR, helicase region; NC, N-terminal cassette. Values represent means \u00b1 SD; n = 3 technical replicates. Apparent ATPase rates were calculated as described in the Methods and in Supplementary Fig. 4. Significance indicators represent the significance of differences to wt ASCC3HR; ns, not significant; ****, p \u2264 0.0001. ASCC3HR constructs, in which either the NC (D611A) or the CC (D1463A) are inactivated show reduced ATPase activities, and ASC1 does not significantly enhance the ASCC3HR ATPase.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_5.jpeg",
+        "caption": "Path of DNA through the ASCC3HR-ASC1 complex.\na, Orthogonal, semitransparent views of an electrostatic surface representation of the ASCC3HR-ASC1 complex with superimposed DNA (gold), modeled according to DNA binding by the Hel308 DNA helicase. Red, negative charge; blue, positive charge.\nb, SDS-PAGE analysis monitoring UV-induced cross-linking of radio-labeled oligo-T DNAs (indicated at the bottom) to ASCC3HR (lanes 2, 3, 7, 8, 12, 13, 17, 18) or to the ASCC3HR-ASC1 complex (lanes 4, 5, 9, 10, 14, 15, 19, 20). Lanes 1, 6, 11, 16, DNAs alone. Numbers above the gel indicate the amounts of ASCC3HR and ASC1 (1, 100 nM; 2, 200 nM) added to 4.3 nM radio-labeled DNA. Labeled bands are identified on the right.\nc, Quantification of the data in (b) obtained with samples containing 200 nM ASCC3HR or ASCC3HR-ASC1. Bars represent means \u00b1 SD; n = 3 technical replicates. Individual data points are shown as spheres.\nd, Semi-transparent surface view of the ASCC3HR-ASC1 complex (ASCC3HR, light gray; ASC1, dark gray) with part of the ASC1 ZnF-lasso linker region (violet) according to an AlphaFold24 model of ASC1. DNA (red) modeled according to DNA binding by the Hel308 DNA helicase is shown as a cartoon. Cross-linked residues (ASCC3HR NC, blue; ASCC3HRCC, cyan) and a cross-linked peptide (ASC1, green) as identified by MS are shown as spheres, lining the putative path of the ssDNA region through both cassettes and exiting the CC near the ASC1 ASCH domain.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_6.jpeg",
+        "caption": "ASC1 and ALKBH3 may conscribe ASCC core subunits for distinct cellular functions.\na, SDS-PAGE analyses of analytical SEC elution fractions monitoring the competitive binding of ASC1 and AlkBH3 to ASCC3HR. Throughout all panels, equivalent elution fractions are vertically aligned. Input samples are identified on top of each run. Molecular mass markers in kDa are shown on the left; protein bands are identified on the right. Stable complexes eluting from some analytical SEC runs are identified below the respective gels. For some analytical SEC runs, separate regions of the same gel were spliced together for display purposes (see Source Data file for uncropped gels). Dashed lines, splice lines. ASC1 and AlkBH3 do not stably interact (run 4). AlkBH3 and ASC1 form stable binary complexes with ASCC3HR (runs 5 and 6). AlkBH3 is excluded from a pre-formed ASCC3HR-ASC1 complex (run 7).\nb, Western blots documenting CRISPR/Cas9-mediated KO of ASC1. GAPDH was used as a loading control.\nc, Assay comparing the relative degree of viability of ASC1 wt and KO PC-3 cells in the presence of increasing concentrations of MMS. ASC1 wt cells, black; ASC1 KO cells, red. Values represent means \u00b1 SD; n = 5 technical replicates. Error bars are hidden by data points.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    }
+]

059a54d253ab5af79d48ff1a3f02886f0511ffb2444cb09d7b8ed598ea46c8cb/preprint/preprint.md

@@ -0,0 +1,243 @@
+# Abstract
+
+Activating signal co-integrator complex (ASCC) supports diverse genome maintenance and gene expression processes. Its ASCC3 subunit is an unconventional nucleic acid helicase, harboring tandem Ski2-like NTPase/helicase cassettes crucial for ASCC functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. Here, we present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as *in vitro* and cellular functional analyses of the ASCC3-ASC1/TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. ASC1 docks on ASCC3 *via* a zinc finger domain and stimulates the helicase by positioning a C-terminal ASC1-homology domain next to the C-terminal helicase cassette of ASCC3, likely assisting the DNA exit. ASC1 binds ASCC3 mutually exclusively with the DNA/RNA dealkylase, ALKBH3, directing ASCC for specific processes. Our findings define ASCC3-ASC1/TRIP4 as a tunable motor module of ASCC that encompasses two cooperating ATPase/helicase units functionally expanded by ASC1/TRIP4.
+
+# Introduction
+
+Human activating signal co-integrator complex (ASCC) has been implicated in a surprisingly diverse range of genome maintenance and gene expression processes, including transcriptional regulation, DNA repair and ribosome quality control. ASCC was originally described to comprise four subunits, i.e., activating signal co-integrator 1/thyroid receptor-interacting protein 4 (ASC1/TRIP4; “ASC1” in the following), ASCC1, ASCC2 and ASCC3. However, different sets of ASCC subunits have been implicated in different ASCC-dependent processes, suggesting that ASCC’s subunit composition or requirements may differ for different cellular functions. By associating with basal transcription factors, nuclear receptors and/or various co-activators, ASCC is thought to establish distinct transcription co-activator complexes in response to different cellular conditions. Moreover, the ASCC3 subunit has been identified as a modulator of antiviral type I interferon-stimulated genes during infections by positive-strand RNA viruses. ASCC2 and, in particular, ASCC3 have also been implicated in the suppression of long mRNA isoforms, due to a decrease in transcription elongation rates and instigation of alternative last exon splicing, upon UV irradiation or exposure to agents that give rise to bulky DNA lesions; a short ascc3 transcript, itself originating from alternative last exon splicing, in turn acts as a long non-coding RNA during transcriptional recovery.
+
+ASCC3, supported by ASC1, ASCC1 and ASCC2, is also involved in ribosome and translation quality control pathways. In contrast, only ASCC1, ASCC2 and ASCC3, have additionally been found to be important for DNA alkylation damage repair, for which the factors associate with the single-stranded (ss) DNA/RNA-specific α-ketoglutarate/iron-dependent dioxygenase, ALKBH3; ASC1 has not yet been implicated in DNA alkylation damage repair. Finally, ASCC, possibly in different constellations, may also help mediate RNA modification/repair processes. For example, a proteomics analysis suggested that ASC1, ASCC1, ASCC2 and ASCC3 interact with ZCCHC4, a methyl-transferase that introduces a m⁶A modification at position A4220 of 28S rRNA. Furthermore, ASCC3 is required for efficient, ALKBH3-dependent removal of m¹A and m³C modifications from mRNAs, and for alkylation-induced P-body formation.
+
+ASCC3 contributes to all of the above ASCC-related functions. It is a large nucleic acid-dependent NTPase that can act as a 3’-to-5’ translocase/helicase. NTPase-fueled remodeling of nucleic acids or nucleic acid-protein complexes by ASCC3, therefore, likely constitute central activities for all of ASCC’s diverse cellular roles. For example, during DNA alkylation damage repair, ASCC3 generates single-stranded DNA for dealkylation by ALKBH3. Furthermore, mutations in conserved NTPase/helicase motifs of ASCC3 interfere with ASCC-mediated splitting of stalled ribosomes during ribosome or translation quality control, and ASCC3 has been suggested to disassemble ribosomes collided on alkylated mRNAs for dealkylation by ALKBH3.
+
+ASCC3 is an unconventional nucleic acid-dependent NTPase that is closely related to the spliceosomal RNA helicase, U5 small nuclear ribonucleoprotein 200 kDa (SNRNP200/BRR2). ASCC3 and SNRNP200 contain a tandem array of Ski2-like helicase cassettes (N-terminal cassette, NC; C-terminal cassette, CC), preceded by ~400-residue N-terminal regions that can auto-inhibit the helicase activities. In SNRNP200, only the NC is an active NTPase and helicase, while the CC acts as an intra-molecular helicase co-factor. In contrast, both helicase cassettes in ASCC3 may be enzymatically active. However, presently the molecular mechanisms underlying ASCC3 nucleic acid translocase/helicase activities and its regulation are poorly understood.
+
+Here, we find a hitherto unobserved mechanism of nucleic acid translocation/unwinding in ASCC3 and reveal that it is regulated by ASC1. Using cryogenic electron microscopy (cryoEM)/single-particle analysis (SPA) and DNA-protein cross-linking/mass spectrometry (CLMS)-based structural analyses as well as systematic protein interaction, DNA binding and unwinding assays, we show that ASCC3 can thread DNA through both of its helicase cassettes. ASC1 docks to the ASCC3 CC via a zinc finger (ZnF) domain, positioning its ASC1-homology (ASCH) domain such that it can engage DNA exiting from ASCC3. We also present evidence that ASC1 and ALKBH3 engage ASCC3 in a mutually exclusive manner and that ASC1 does not affect ASCC-dependent DNA alkylation damage repair, suggesting that ASC1 and ALKBH3 are facultative, process-specific ASCC subunits or auxiliary proteins.
+
+# Results
+
+ASCC1 and ASCC2 directly interact with ASCC3, suggesting that ASCC3 forms the main scaffold for the ASCC and possibly an interaction platform for ASCC-auxiliary proteins. We therefore tested whether ASC1 also directly binds ASCC3 *in vitro*. While ASC1 did not stably interact with the ASCC3 N-terminal region (ASCC3<sup>NTR</sup>, residues 1–400), it co-eluted with the helicase region of ASCC3 (ASCC3<sup>HR</sup>, residues 401–2202) in analytical size-exclusion chromatography (SEC; Fig. <span class="InternalRef">1</span> a,b).
+
+Next, we reconstituted an ASCC3<sup>HR</sup>-ASC1 complex and determined its atomic structure *via* cryoEM/SPA at a nominal resolution of 3.4 Å (Fig. <span class="InternalRef">2</span> a; Supplementary Fig. 1; Supplementary Fig. 2; Supplementary Table 1). In the cryoEM reconstruction, we could trace residues 401–2183 of ASCC3<sup>HR</sup> as well as residues 168–219 and 375–580 of ASC1 (Fig. <span class="InternalRef">2</span> b,c), capitalizing on AlphaFold-predicted models <sup><span class="CitationRef">24</span></sup>. ASCC3<sup>HR</sup> adopts a structure very similar to the helicase region of SNRNP200 (root mean square deviation [rmsd] of 3.1 Å for 1,504 pairs of Cα atoms compared to isolated SNRNP200<sup>HR</sup>; PDB ID 4F91; Supplementary Fig. 3) <sup><span class="CitationRef">21</span></sup>. Like SNRNP200, both ASCC3 helicase cassettes contain consecutive dual RecA-like (RecA1, RecA2), winged-helix (WH), helical bundle (HB), helix-loop-helix (HLH) and immunoglobulin-like (IG) domains and associate to form a compact helicase region (Fig. <span class="InternalRef">2</span> c). An extended, irregularly structured linker (residue 1296–1306) connects the IG domain of the NC to the RecA1 domain of the CC, running closely along the body of the ASCC3 CC (Fig. <span class="InternalRef">2</span> a–c).
+
+ASC1 exclusively associates with the CC of ASCC3<sup>HR</sup> (Fig. <span class="InternalRef">2</span> a,b). Residues 168–219 of ASC1 fold into a dual-ZnF domain, with residues C171/C173/H178/C192 and C184/C187/C200/C203 each coordinating a zinc ion (Fig. <span class="InternalRef">2</span> b). The ZnF domain of ASC1 rests on top of the RecA1 domain of the ASCC3 CC, neighboring the extended linker to the NC (Fig. <span class="InternalRef">2</span> b) and spanning ~757 Å<sup><span class="CitationRef">2</span></sup> of interface area, with hydrophobic interactions in the center and hydrophilic interactions at the periphery (Fig. <span class="InternalRef">2</span> d, left). ASC1 residues 375–424 lack a globular fold and regular secondary structure elements, except for a short helical region in residues 398–405. They form a lasso-like structure around a protruding edge of the C-terminal ASCC3 WH domain (Fig. <span class="InternalRef">2</span> b; Fig. <span class="InternalRef">2</span> d, middle), with residues 411–424 inserted deeply into a groove between the RecA1, WH, HB and IG domains of the ASCC3<sup>HR</sup> CC, spanning ~1,914 Å<sup><span class="CitationRef">2</span></sup> of interface area with ASCC3<sup>HR</sup>. ASC1 residues 411–424 form a support for the C-terminal ASCH domain of ASC1 (residues 425–578) that further interconnects the C-terminal ASCC3 RecA1, WH and IG domains (Fig. <span class="InternalRef">2</span> b; Fig. <span class="InternalRef">2</span> d, right), spanning an additional ~1,321 Å<sup><span class="CitationRef">2</span></sup> of interface area with ASCC3<sup>HR</sup>.
+
+**The ZnF domain is required for stable docking of ASC1 on ASCC3<sup>HR</sup> *in vitro***
+
+Based on the structure, we designed various ASC1 fragments to probe the importance of different regions for stable complex formation with ASCC3<sup>HR</sup>. Consistent with the cryoEM structure, the N-terminal 80 residues of ASC1 did not sustain a stable interaction with ASCC3<sup>HR</sup> (Fig. <span class="InternalRef">1</span> c), while ASC1 residues 152–581, encompassing the ZnF domain, the lasso-like peptide and the ASCH domain, co-migrated with ASCC3<sup>HR</sup> in analytical SEC (Fig. <span class="InternalRef">1</span> d). An N-terminal ASC1 region including the ZnF domain (residues 1–230) or the ZnF domain alone (residues 152–230) also stably bound ASCC3<sup>HR</sup> (Fig. <span class="InternalRef">1</span> e). In contrast, C-terminal ASC1 residues 281–403, 403–581 or 281–581, containing the lasso-like peptide, the ASCH domain or both, did not support stable complex formation with ASCC3<sup>HR</sup> (Fig. <span class="InternalRef">1</span> f), although these regions span a considerably larger interface with ASCC3<sup>HR</sup> than the ZnF domain (see above). Thus, only the ZnF domain of ASC1 is required for stable complex formation *in vitro*, and only upon anchoring *via* the ZnF domain, the C-terminal ASCH domain and the preceding peptide region of ASC1 are stably docked on the ASCC3 CC.
+
+**Cellular interaction tests corroborate *in vitro* interaction patterns**
+
+To test the importance of ASC1 regions for the interaction with ASCC3 and other ASCC subunits in cells, we generated stably transfected Flp-In™ T-REx™ 293 cell lines for the inducible expression of N- or C-terminally Flag-tagged versions of full-length ASC1 or truncation variants lacking either N-terminal regions including the ZnF domain (ASC1<sup>Δ1−276</sup>) or lacking the C-terminal ASCH domain (ASC1<sup>Δ403−581</sup>). Immuno-fluorescence microscopy showed that all constructs were located to both the cytosol and the nucleus (Fig. <span class="InternalRef">3</span> a). We then immuno-precipitated the Flag-tagged ASC1 variants with α-Flag antibodies and probed the eluates for the presence of other ASCC subunits by western blot. Irrespective of the position of the tag, ASC1 and ASC1<sup>Δ403−581</sup> (lacking the ASCH domain) co-precipitated ASCC1, ASCC2 and ASCC3 (Fig. <span class="InternalRef">3</span> b). In contrast, no interaction with these ASCC subunits was detected by co-precipitation with ASC1<sup>Δ1−276</sup> (lacking the ZnF domain; Fig. <span class="InternalRef">3</span> b).
+
+To further test the relevance of ASCC3<sup>HR</sup>-ASC1 contacts observed in our cryoEM structure for the interaction of ASCC3 and ASC1 in cells, we transfected 293T cells for the expression of N-terminally HA-tagged versions of ASC1. In these ASC1 variants, either the ZnF domain was precisely deleted (ΔZnF; deletion of residues 168–219), three residues that engage in hydrophobic interactions with ASCC3<sup>HR</sup> were exchanged for alanines (LLI-AAA, ASC1<sup>L174A−L180A−I190A</sup>; Fig. <span class="InternalRef">2</span> d, left) or two cysteines coordinating the first (C171) and second (C184) Zn<sup>2+</sup> ion were exchanged for alanines (CC-AA, ASC1<sup>C171A−C184A</sup>). While wild type (wt) ASC1 efficiently co-immuno-precipitated endogenous ASCC3, the ΔZnF and CC-AA variants entirely lost the ability to immuno-precipitate ASCC3, and the ASCC3 interaction of the LLI-AAA variant was strongly reduced (Fig. <span class="InternalRef">3</span> c).
+
+Together, the results of these cellular interaction studies are fully in line with the *in vitro* ASCC3<sup>HR</sup>-ASC1 interaction profiles. They confirm that the ZnF domain of ASC1 is the main ASCC3-interacting domain of ASC1, *via* which ASC1 also seems to be incorporated into the ASCC, and suggest that ASC1, ASCC1 and ASCC2 can concomitantly interact with ASCC3. The observations also confirm that our cryoEM structure closely represents the mode of interaction of ASCC3 and ASC1 in cells.
+
+## ASC1 activates ASCC3 helicase activity without influencing ASCC3 ATPase activity
+
+To test the effect of ASC1 on the helicase activity of ASCC3<sup>HR</sup>, we conducted fluorescence-based unwinding assays in a stopped-flow device (Fig. <span class="InternalRef">4</span> a). As this assay tested multiple rounds of unwinding, the observed time traces were fit to a double exponential equation, and amplitude-weighted unwinding rate constants (*k*<sub><em>uaw</em></sub>) were calculated for the comparison of unwinding efficiencies. <sup><span class="CitationRef">25</span>–<span class="CitationRef">27</span></sup> ASCC3<sup>HR</sup> alone efficiently unwound the substrate DNA (*k*<sub><em>uaw</em></sub> = 0.024 s<sup>−1</sup>), but unwinding was further stimulated 2.3-fold by ASC1 (*k*<sub><em>uaw</em></sub> = 0.054 s<sup>−1</sup>; Fig. <span class="InternalRef">4</span> b; Supplementary Table 2). In contrast, both ASC1<sup>1–230</sup> (encompassing the ZnF domain), which stably bound ASCC3<sup>HR</sup> in analytical SEC, as well as ASC1<sup>403–581</sup> (encompassing the ASCH domain and preceding peptide), which did not co-migrate with ASCC3<sup>HR</sup> in analytical SEC, only marginally affected the ASCC3<sup>HR</sup> helicase activity (*k*<sub><em>uaw</em></sub> = 0.030 s<sup>−1</sup> and 0.035 s<sup>−1</sup>, respectively; Fig. <span class="InternalRef">4</span> b; Supplementary Table 2). Thus, while the ASC1 ZnF domain alone can stably bind to ASCC3<sup>HR</sup>, it does not efficiently activate ASCC3<sup>HR</sup> helicase activity, for which the lasso-like peptide and ASCH domain are also required.
+
+Next, we asked which helicase cassette of ASCC3<sup>HR</sup> is preferentially regulated by ASC1. To this end, we employed ASCC3<sup>HR</sup> variants, in which a crucial motif II aspartate of the NC (D611) or CC (D1453) was exchanged for an alanine (Fig. <span class="InternalRef">4</span> c), abrogating NTPase/helicase activity in the respective cassette. <sup><span class="CitationRef">20</span></sup> ASCC3<sup>HR,D1453A</sup>, bearing an inactive CC, unwound DNA at a reduced rate (*k*<sub><em>uaw</em></sub> = 0.011 s<sup>−1</sup>), while the unwinding activity of ASCC3<sup>HR,D611A</sup>, containing an inactive NC, was strongly reduced (*k*<sub><em>uaw</em></sub> n.d.; Fig. <span class="InternalRef">4</span> d), suggesting that both cassettes are required for full ASCC3 helicase activity. Only the construct bearing an inactive CC was stimulated by ASC1 to quantifiable levels (ASCC3<sup>HR,D1453A</sup>-ASC1 *k*<sub><em>unw</em></sub> = 0.024 s<sup>−1</sup>; Fig. <span class="InternalRef">4</span> d; Supplementary Table 2).
+
+NTPase activity associated with both ASCC3<sup>HR</sup> cassettes was further corroborated by DNA-stimulated ATPase assays. ASCC3<sup>HR,D1453A</sup> (inactive CC) and ASCC3<sup>HR,D611A</sup> (inactive NC) exhibited ~28 and ~73% of the DNA-stimulated ATPase activity of wt ASCC3<sup>HR</sup>, while the DNA-stimulated ATPase activity of the ASCC3<sup>HR,DD611/1453AA</sup> variant, with motif II changes in both cassettes, was negligible (Fig. <span class="InternalRef">4</span> e; Supplementary Fig. 4). As expected if the implemented residue exchanges selectively abrogated ATPase activity in the respective cassette, the ATPase activity of ASCC3<sup>HR,D1453A</sup> (inactive CC) closely matched the ATPase activity of the isolated wt NC (Fig. <span class="InternalRef">4</span> e; Supplementary Fig. 4). As we failed to produce the ASCC3 CC in isolation, a similar comparison could not be drawn between ASCC3<sup>HR,D611A</sup> (inactive NC) and isolated wt CC. Irrespectively, in contrast to the helicase activity, the stimulated ATPase activity of ASCC3<sup>HR</sup> was not further enhanced by ASC1 (Fig. <span class="InternalRef">4</span> e). Thus, ASC1 activates ASCC3<sup>HR</sup> helicase activity without affecting its ATPase activity.
+
+## DNA can be threaded through both ASCC3 helicase cassettes and along ASC1
+
+We failed to obtain cryoEM structures of ASCC3<sup>HR</sup> or ASCC3<sup>HR</sup>-ASC1 in complex with ssDNA or with dsDNA bearing a 3’-ss overhang. Modeling of putative ssDNA binding to the NC and CC of ASCC3<sup>HR</sup> by superimposing a structure of the Hel308 DNA helicase in complex with DNA (PDB ID 2P6R) <sup><span class="CitationRef">28</span></sup> on both ASCC3<sup>HR</sup> cassettes indicated that ssDNA could be threaded consecutively through both helicase cassettes and might exit the CC close to the ASC1 ASCH domain (Fig. <span class="InternalRef">5</span> a). Positive electrostatic surface potential is in agreement with the modeled path of ssDNA, in particular for the ASCC3<sup>HR</sup> NC (Fig. <span class="InternalRef">5</span> a). The model suggested that a minimum of 24 nucleotides (nts) of ssDNA are required to traverse the two cassettes and ASC1. In contrast, lateral entry of ssDNA to the CC, circumventing the NC, is blocked in the conformation of ASCC3<sup>HR</sup> observed in our cryoEM structure. A requirement for DNA to enter the CC *via* the preceding NC would be consistent with the larger effect on helicase activity we observed upon inactivating the NC alone as compared to a ASCC3<sup>HR</sup> variant containing only an inactive CC (see Fig. <span class="InternalRef">4</span>).
+
+To test if, during unwinding, ASCC3<sup>HR</sup> and ASCC3<sup>HR</sup>-ASC1 might thread single-stranded DNA through both helicase cassettes, and in the latter case along the ASC1 ASCH domain, we conducted ultra-violet (UV) irradiation-induced cross-linking of ASCC3<sup>HR</sup> and ASCC3<sup>HR</sup>-ASC1 to variable-length, single-stranded oligo-T DNAs (T<sub>12</sub>, T<sub>24</sub>, T<sub>36</sub>, T<sub>48</sub>; Fig. <span class="InternalRef">5</span> b). Both ASCC3<sup>HR</sup> and ASCC3<sup>HR</sup>-ASC1 did not efficiently cross-link to T<sub>12</sub> ssDNA and showed stepwise increased cross-linking to T<sub>24</sub>, T<sub>36</sub> and T<sub>48</sub> DNAs (cross-link efficiencies of ~30%, 80% and 90%, respectively; Fig. <span class="InternalRef">5</span> b,c). ASC1 alone did not efficiently cross-link to any of the DNA samples. These observations are consistent with the notion that a ssDNA region sufficiently long to traverse both cassettes is required for DNA to be efficiently engaged by ASCC3<sup>HR</sup> or ASCC3<sup>HR</sup>-ASC1.
+
+Next, we subjected ASCC3<sup>HR</sup> or ASCC3<sup>HR</sup>-ASC1 after UV-induced cross-linking to T<sub>48</sub> ssDNA to DNase/protease digestion followed by mass spectrometric analysis of cross-linked peptide-DNA conjugates. We observed one cross-linked peptide each in ASC1 (region connecting ZnF and lasso), the RecA1 domain of the ASCC3<sup>HR</sup> NC (corresponding to helicase motif Ia), the N-terminal WH domain and the C-terminal WH domain, as well as two cross-linked peptides in the CC IG domain (Table <span class="InternalRef">1</span>). With exception of the ASC1 peptide, we could identify one or two specific cross-linked residues in these peptides (Table <span class="InternalRef">1</span>; RecA1<sup>NC</sup>, M546; WH<sup>NC</sup>, Y988; WH<sup>CC</sup>, Y1821 and Y1822; IG<sup>CC</sup>, C2101 and Y2135). The cross-linked residues and the modeled cross-linked ASC1 peptide are positioned closely along the path of the modeled DNA (Fig. <span class="InternalRef">5</span> d). Together, these observations are consistent with the idea that during unwinding, ssDNA is threaded through both helicase cassettes and along ASC1 in the vicinity of the ASCH domain. It is, however, also possible that ASCC3<sup>HR</sup> may undergo conformational changes upon binding to ssDNA of sufficient length, so that the substrate can engage the NC and CC independently.
+
+**Table 1: DNA-protein cross-links identified by MS.**
+
+| Cross-linked peptide<sup>1</sup> | Cross-linked residue | Trial | Domain or region | Motif<sup>2</sup> |
+|----------------------------------|------------------------|-------|------------------|-------------------|
+| 257-SGLEK-261 | n.i.<sup>3</sup> | 1 | ZnF-lasso linker | - |
+| 541-ALAAE<u><span style="color:blue">M</span></u>TDYFSR-552 | M546 | 2 | RecA1<sup>NC</sup> | Ia |
+| 984-TASH<u><span style="color:blue">Y</span></u>YIK-991 | Y988 | 1,2 | WH<sup>NC</sup> | - |
+| 1818-IAS<u><span style="color:#33CCFF">Y</span></u>YLK-1825 | Y1821 | 2 | WH<sup>CC</sup> | - |
+| 1818-IASY<u><span style="color:#33CCFF">Y</span></u>LK-1825 | Y1822 | 2 | WH<sup>CC</sup> | - |
+| 2096-GKPES<u><span style="color:#33CCFF">C</span></u>AVTPR-2106 | C2101 | 2 | IG<sup>CC</sup> | - |
+| 2133-VG<u><span style="color:#33CCFF">Y</span></u>IR-2137 | Y2135 | 1,2 | IG<sup>CC</sup> | - |
+
+<sup>1</sup> Cross-linked residue(s) colored as in (c) and underlined  
+<sup>2</sup> NC helicase motif Ia, residues 536–546  
+<sup>3</sup> n.i., not identified
+
+## ASC1 and ALKBH3 support ASCC core subunits in distinct cellular functions
+
+Present data suggest that ASCC core subunits may associate with different auxiliary proteins to participate in distinct genome maintenance and gene expression processes. More specifically, ASC1 has so far been found associated with ASCC-dependent transcription regulation <sup><span class="CitationRef">1</span>, <span class="CitationRef">2</span>, <span class="CitationRef">4</span>, <span class="CitationRef">5</span></sup> and ribosome quality control <sup><span class="CitationRef">9</span>, <span class="CitationRef">11</span>, <span class="CitationRef">12</span></sup>, while ALKBH3 is associated with ASCC3 during DNA dealkylation repair <sup><span class="CitationRef">13</span>, <span class="CitationRef">14</span></sup>. We therefore wondered whether ASC1 and ALKBH3 might bind ASCC3 in a mutually exclusive manner. To test this notion, we conducted competitive SEC-based interaction studies. ASC1 and ALKBH3 did not co-migrate during SEC (Fig. <span class="InternalRef">6</span> a). A portion of ALKBH3 stably associated with ASCC3<sup>HR</sup> in SEC, but failed to be incorporated into a pre-formed ASCC3<sup>HR</sup>-ASC1 complex (Fig. <span class="InternalRef">6</span> a). These findings suggest that ASC1 and ALKBH3 engage ASCC3<sup>HR</sup> in a mutually exclusive manner, possibly by taking advantage of overlapping binding sites, and that ASC1 might associate more strongly with ASCC3<sup>HR</sup> than ALKBH3.
+
+To further test the idea that either ASC1 or ALKBH3 associates with ASCC core subunits depending on the particular ASCC-dependent cellular process, we explored the effect of ASC1 on DNA dealkylation damage repair, where ALKBH3 is known to be involved. To this end, we knocked out ASC1 *via* CRISPR/Cas9-based genome engineering in human PC-3 cells (Fig. <span class="InternalRef">6</span> b) and tested the response of the edited and parental cells to methyl methanesulfonate (MMS) treatment. ASC1 knockout (KO) did not impact cell survival in the presence of even high concentrations of MMS (Fig. <span class="InternalRef">6</span> c), suggesting that ASC1 may not be involved in ASCC3/ALKBH3-mediated DNA dealkylation <sup><span class="CitationRef">13</span></sup>. Together, these observations suggest that ASC1 represents a process-specific ASCC subunit that regulates ASCC3 helicase activity during ASCC-dependent transcriptional events and ribosome rescue, but may be replaced by ALKBH3 during ASCC-dependent DNA dealkylation damage repair.
+
+# Discussion
+
+ASCC is a multi-functional complex. While apparently different sets of ASCC core and auxiliary factors participate in different ASCC-dependent processes, the large nucleic acid helicase, ASCC3, seems to provide crucial molecular motor activity for all of ASCC’s multiple functions. ASCC3 has striking homology to the spliceosomal RNA helicase, SNRNP200, and the two proteins represent the only known human members of a unique sub-family of Ski2-like helicases that possess tandem helicase cassettes. In SNRNP200, only the NC is an active ATPase/RNA helicase, while the CC acts as an intra-molecular modulator of the NC helicase.¹⁹,²⁹
+
+Here, we show by cryoEM-based structural analysis that ASCC3 indeed contains a dual-cassette helicase region that closely resembles the analogous region of SNRNP200, at least in the absence of factors other than ASC1. In line with previous observations⁹–¹¹,¹³,²⁰, our systematic DNA unwinding and ATPase assays strongly suggest that, in contrast to SNRNP200, both ASCC3 cassettes are active ATPases and helicases. Our DNA-protein CLMS analyses are consistent with a model in which ASCC3 translocates relative to ssDNA during DNA unwinding, threading one DNA strand consecutively through both helicase units. In principle, our data would also be consistent with the two ASCC3 helicase cassettes unwinding DNA independently of each other. However, in the ASCC3 HR conformation observed here, direct accommodation of ssDNA at the CC is blocked by the NC. Thus, for the latter scenario, ASCC3 would have to undergo a large conformational rearrangement that leads to a separation of its helicase cassettes if ssDNA were to be captured by the CC without being first threaded through the NC. As ASCC3 interacts with different substrate complexes and auxiliary proteins in different functional contexts, which could provoke conformational changes in ASCC3, it is conceivable that in certain scenarios the helicase activity of either individual cassette is employed, while in others the two helicase cassettes operate in tandem. Furthermore, in a given functional scenario the two cassettes may even translocate the same or different nucleic acid molecules (see also below).
+
+The CC of SNRNP200 serves as an interaction platform for numerous proteins, several of which inhibit its NC helicase activity from a distance³⁰–³². In contrast, the C-terminal Jab1 domain of the large spliceosomal PRPF8 scaffold that can activate the SNRNP200 helicase directly binds the active NC.³³,³⁴ Here, we find that similar to the situation in SNRNP200, the ASCC3 CC serves as a binding platform for the ASC1 protein. ASC1 predominantly latches onto ASCC3 via its ZnF domain, allowing the positioning of an ASCH domain close to the presumed DNA exit of the ASCC3 CC with the help of the intervening lasso peptide. However, unlike many proteins that bind the SNRNP200 CC, we show that ASC1 stimulates ASCC3 helicase activity. The ZnF docking domain is insufficient for helicase stimulation, which also requires C-terminal ASC1 regions including the ASCH domain. While we cannot yet pinpoint the precise molecular mechanism, by which ASC1 stimulates ASCC3, our DNA-protein CLMS data support the notion that the ASCH domain or neighboring regions may facilitate DNA exit from the ASCC3 CC. Indeed, the ASCH domain belongs to a large family of domains that bind nucleic acids.³⁵,³⁶
+
+Cooperation between both helicase cassettes and activation of ASCC3 helicase activity by ASC1 may be required to unfold sufficiently strong or appropriately coordinated motor activity during transcription regulatory processes and ribosome quality control, where both ASCC3 and ASC1 are involved. While the targets of ASCC3’s motor activity during transcriptional regulation are presently unknown, during ribosome quality control, ASCC3’s ATP-dependent motor activity is essential for the disassembly of the lead ribosome in collided di-somes or polysomes into ribosomal subunits.⁹,¹¹,¹² As no DNA is involved in this process, ASCC3 most likely operates by engaging and translocating mRNA or rRNA regions. Indeed, we know that ASCC3 can also unwind RNA duplexes in vitro, suggesting that it is also an RNA translocase, but its RNA helicase/translocase activity is much less efficient than its DNA helicase/translocase activity (unpublished). Our analyses show that inactivation of either ASCC3 cassette leads to partial loss of ASCC3 helicase activity. Thus, splitting of ribosomes by translocating on a sub-optimal mRNA or rRNA substrate may require (a) ASCC3 resorting to a translocation mode that involves both active cassettes on the same or on different RNA molecules, (b) additional stimulation by ASC1 and/or (c) stimulation by another accessory factor that promotes ASCC3 RNA translocase/helicase function.
+
+Recent cryoEM structures of yeast ribosome quality control trigger complex (RQT)-ribosome complexes revealed that prior to ribosome splitting the yeast ASCC3 ortholog, Slh1p, can adopt a more open conformation with fewer direct interactions between the two helicase cassettes as observed in our human ASCC3-ASC1 complex structure.³⁷ While in the imaged conformations both Slh1p helicase cassettes are potentially accessible to an RNA substrate, no corresponding substrate density was observed at either Slh1p cassette.³⁷ In the observed conformations, mRNA could apparently be accommodated directly at the Slh1p CC, but an Slh1p variant harboring an ATPase-deficient NC (Slh1p K361R) was required to capture RQT-ribosome complexes at a stage preceding ribosome splitting³⁷, indicating that the NC ATPase/helicase activity is also required for the splitting reaction. Thus, whether both cassettes or only one of them translocate mRNA or whether one cassette engages mRNA while the other operates on an rRNA region during ribosome splitting remains to be elucidated.
+
+Findings reported here also underscore the notion that ASCC exhibits compositional dynamics that allow it to participate in different processes. We find that ASC1, which collaborates with ASCC3 during transcriptional and ribosome quality control, binds to ASCC3 in a manner that is mutually exclusive to ALKBH3, which capitalizes on the ASCC3 helicase activity during DNA alkylation damage repair. Consistent with the idea of these two factors associating with ASCC3 in different functional scenarios, we also show that ASC1 does not impact cell sensitivity to an alkylating agent, unlike ALKBH3 or other subunits of the ASCC complex.¹³,¹⁵ As ASC1 seems to associate more stably with ASCC3 HR than ALKBH3, it remains to be seen if additional factors may aid ALKBH3 in displacing ASC1 for DNA dealkylation damage repair. Additional interactors may favor a conformation of ASCC3 that exhibits altered ALKBH3 affinity. It is also possible, that the protein interactions of ASCC3 may be dynamically regulated by specific post-translational modifications or by the recruitment of subsets of factors to specific sub-cellular compartments. Both of the latter principles have been shown to play a role during ASCC-related cellular processes.⁹,¹⁰,¹⁴,¹⁵,¹⁹,³⁸,³⁹
+
+# Methods
+
+## Molecular cloning
+DNA fragments encoding ASCC3<sup>HR</sup> (wt, D611A, D1453A or D611A-D1453A) or ASCC3<sup>NC</sup> were cloned into a pFL vector for expression as N-terminally His<sub>10</sub>-tagged, TEV-cleavable proteins via recombinant baculoviruses in insect cells as described previously.<sup>20</sup> A DNA fragment encoding full-length (FL) ASC1 was PCR-amplified from a synthetic gene (IDT; Supplementary Table 3) and inserted into the pETM-11 or pIDS vectors (EMBL, Heidelberg) for expression as an N-terminally His<sub>6</sub>-tagged, TEV-cleavable protein. See Supplementary Table 4 for PCR primers used. The pIDS-<em>asc1</em><sup>FL</sup> construct was Cre-recombined with pFL-<em>ascc3</em><sup>HR</sup> for co-expression via a recombinant baculovirus in insect cells. DNA fragments encoding ASC1<sup>1–80</sup>, ASC1<sup>1–230</sup>, ASC1<sup>152–230</sup>, ASC1<sup>152–581</sup>, ASC1<sup>281–403</sup>, ASC-1<sup>281–581</sup> or ASC-1<sup>403–581</sup> were amplified via PCR from the pETM-11-<em>asc1</em><sup>FL</sup>, and re-cloned into the pETM-11 vector. A DNA fragment encoding full-length ALKBH3 was PCR-amplified from a cDNA library of human HeLa cells and inserted into the pETM-11 vector for expression as an N-terminally His<sub>6</sub>-tagged, TEV-cleavable protein. All constructs were verified by Sanger sequencing.
+
+For the preparation of an ASC1 sgRNA vector, we followed a previously established method<sup>40</sup>, cloning the target sequence into the pLenti-CRISPRV2 vector<sup>41</sup>. Primers used for generating the DNA fragment containing the target sequence is shown in Supplementary Table 4.
+
+For expression of HA-tagged ASC1 variants, DNA fragments encoding wt or ΔZnF ASC1 were cloned into pENTR-3C using a synthetic gene (IDT; Supplementary Table 3). Vectors encoding HA-tagged variants ASC1<sup>L174A−L180A−I190A</sup> or ASC1<sup>C171A−C184A</sup> were created using the In-Fusion Snap Assembly mutagenesis kit (Takara Bio #683945). Each construct was then cloned into pHAGE-HA-Blast vector<sup>14</sup> via Gateway recombination. All constructs were verified by Sanger sequencing.
+
+## Generation of cell lines
+Stably transfected Flp-In™ T-REx™ 293 cell lines for the tetracycline-inducible expression of ASC1 variants with N-terminal 2xFlag-His<sub>6</sub> or C-terminal His<sub>6</sub>-2xFlag tags were generated according to the manufacturer’s guidelines.<sup>20</sup> Transfection of the parental cell line was done using X-tremeGENE HP DNA Transfection Reagent (Sigma Aldrich). After hygromycin-based selection of cells that had genomically integrated the expression cassette, tetracycline-induced expression of the tagged proteins was confirmed by western blotting using a monoclonal α-Flag M2 antibody (Sigma Aldrich #F3165; 1:7500). For expression of HA-tagged ASC1 variants, the pHAGE-HA-ASC1 vectors encoding HA-tagged ASC1<sup>wt</sup>, ASC1<sup>ΔZnF</sup>, ASC1<sup>L174A−L180A−I190A</sup> or ASC1<sup>C171A−C184A</sup>, were transfected into 293T cells using Transit293 transfection reagent (Mirus Bio).
+
+## CRISPR/Cas9-based genome editing
+The ASC1 sgRNA expression vector was transfected into the Lenti-X 293T cell line (Takara Bio) together with psPAX2 and pCMV-VSVG (Addgene) for lentivirus production. The virus-containing culture medium was collected 72 h post-transfection. Human PC-3 cells were infected with the viral medium and individual clones were selected in 96-well plates. The single KO colonies were analyzed by western blot using an α-ASC1 antibody (sc-376916, Santa Cruz).
+
+## Recombinant protein production and purification
+ASCC3<sup>HR</sup> variants and ASCC3<sup>NC</sup> were produced in High Five cells as described previously.<sup>20</sup> Cell pellets were re-suspended in 20 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 10 mM imidazole, 1 mM DTT, 8.6% (v/v) glycerol (lysis buffer 1), supplemented with cOmplete™ protease inhibitors (Roche) and lysed by sonication using a Sonopuls Ultrasonic Homogenizer HD (Bandelin). The lysate was cleared by centrifugation and filtration. The protein of interest (POI) was captured on Ni<sup>2+</sup>-NTA resin in a gravity flow column, washed with lysis buffer 1 and eluted with lysis buffer 1 containing 400 mM imidazole. Fractions enriched for the POI were supplemented with 1/10 (w/w) TEV protease and dialyzed against 20 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 1 mM DTT, 8.6% (v/v) glycerol (dialysis buffer) overnight. The sample was then diluted to 100 mM NaCl and loaded onto a HiTrap Heparin HP column (Cytiva), pre-equilibrated with lysis buffer 1 containing 100 mM NaCl. After washing with lysis buffer 1 containing 100 mM NaCl, the POI was eluted with a linear gradient to lysis buffer 1 containing 1.5 M NaCl. The fractions containing the POI were pooled and concentrated with a centrifugal concentrator (100 kDa molecular mass cut-off). The concentrated sample was further purified by SEC on a Superdex 200 10/300 GL column (Cytiva) in 20 mM HEPES-NaOH, pH 7.5, 250 mM NaCl, 5% (v/v) glycerol, 1 mM DTT (SEC buffer). Fractions containing the POI were combined, concentrated, aliquoted, flash-frozen in liquid nitrogen and stored at -80°C.
+
+For preparation of the ASCC3<sup>HR</sup>-ASC1<sup>FL</sup> complex, ASC1<sup>FL</sup> was co-produced with ASCC3<sup>HR</sup> in High Five cells. Cell pellets were re-suspended in lysis buffer 1 supplemented with cOmplete™ protease inhibitors, 1 mM DTT and 20 mM imidazole. The samples were lysed by sonication, then the suspension was centrifuged at 56,000 x g for 1 h, the soluble extract was further filtered through 0.8 µM pore size membrane filters (Millipore). The filtered fractions were collected and incubated with Ni<sup>2+</sup>-NTA resin pre-equilibrated with lysis buffer 1 for 2 h with gentle rotation at 4°C. POI-bound resin was loaded on a gravity flow column, washed with lysis buffer 1 and the POI was eluted with lysis buffer 1 containing 400 mM imidazole. To remove the His<sub>6/10</sub>-tags, 1/10 (w/w) of TEV protease was added and the sample was dialyzed against dialysis buffer overnight. Subsequently, the sample was diluted to 50 mM NaCl and loaded on a 5 ml StrepTrap HP column (Cytiva) pre-equilibrated with lysis buffer 1 containing 50 mM NaCl. After washing with lysis buffer 1 containing 50 mM NaCl, the POI was eluted in a linear gradient to lysis buffer 1 containing 1.5 M NaCl. Fractions containing the POI were combined, diluted to 50 mM NaCl, loaded on a 5 ml HiTrap Heparin HP column, washed and eluted in a linear gradient with lysis buffer 1 containing 1.5 M NaCl. Fractions containing the POI were pooled, concentrated and further purified by SEC on a Superdex 200 10/600 GL column (Cytiva) in 20 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 1 mM DTT. Fractions containing the POI were combined, concentrated, aliquoted, flash-frozen in liquid nitrogen and stored at -80°C.
+
+For production of isolated ASC1 variants, the corresponding pETM-11 vectors were transformed into <em>Escherichia coli</em> BL21 (DE3) cells by electroporation for protein production via auto-induction at 18°C.<sup>42</sup> Cells were harvested when cultures reached an optical density (600 nm) of 10. Cell pellets were re-suspended in lysis buffer 1 and supplemented with cOmplete™ protease inhibitors. After sonication, the lysate was cleared by centrifugation. The POI was captured on Ni<sup>2+</sup>-NTA resin in a gravity flow column, washed with lysis buffer 1 and eluted with lysis buffer 1 containing 400 mM imidazole. Fractions enriched for the POI were supplemented with 1/10 (w/w) TEV protease and dialyzed against dialysis buffer overnight. Dialyzed samples were passed through a Ni<sup>2+</sup>-NTA gravity flow column to remove the cleaved His<sub>6</sub>-tag and TEV. For ASC1<sup>FL</sup>, ASC1<sup>152–581</sup>, ASC1<sup>281–581</sup> and ASC1<sup>403–581</sup> fragments, the samples were diluted to 100 mM NaCl, loaded on a HiTrap Heparin HP column, washed and eluted in a linear gradient to lysis buffer 1 containing 1.5 M NaCl. Fractions containing the POI were combined, concentrated and further purified on a Superdex 200 16/600 GL column in SEC buffer.
+
+For purification of the ASC1<sup>1–80</sup>, ASC1<sup>1–230</sup>, ASC1<sup>152–230</sup> and ASC1<sup>281–403</sup> fragments, the Heparin column step was omitted and the final gel filtration was conducted in 20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 1 mM DTT on a HiLoad 16/60 Superdex 75 pg column (Cytiva).
+
+For production of ALKBH3, the corresponding pETM-11 vector was transformed into <em>E. coli</em> C2566 cells by electroporation for protein production via IPTG induction at 37°C. Cell pellets were re-suspended in 20 mM TRIS-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole, 1 mM DTT, 0.1 mM PMSF (lysis buffer 2), and lysed by sonication. The lysate was cleared by centrifugation. The supernatant was loaded onto a Ni<sup>2+</sup>-NTA column, washed with lysis buffer 2 and the POI was eluted with a linear gradient to lysis buffer 2 containing 400 mM imidazole. Fractions enriched for the POI were combined, supplemented with 1/20 (w/w) TEV protease and dialyzed against dialysis buffer overnight. The sample was then diluted to 100 mM NaCl and loaded onto a HiTrap Heparin HP 5 ml column (Cytiva), pre-equilibrated with dialysis buffer containing 100 mM NaCl. After washing with dialysis buffer containing 100 mM NaCl, the POI was eluted with a linear gradient to dialysis buffer containing 1.5 M NaCl. The fractions containing the POI were pooled and concentrated with a centrifugal concentrator (10 kDa molecular mass cut-off). The concentrated sample was further purified by SEC on a Superdex 75 10/60 GL column (Cytiva) in 20 mM TRIS-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT. Fractions containing the POI were combined, concentrated, aliquoted, flash-frozen in liquid nitrogen and stored at -80°C.
+
+## Analytical size exclusion chromatography
+Analytical SEC-based interaction tests were conducted in 20 mM HEPES-NaOH, pH 7.5, 250 mM NaCl, 5% (v/v) glycerol, 1 mM DTT. 100 pmol of ASCC3<sup>HR</sup> were mixed with other proteins in a two to ten-fold molar excess in a final reaction volume of 80 µl. After incubation of the mixtures on ice for 30 min, the samples were loaded on a Superdex 200 3.2/300 analytical size exclusion column (Cytiva). 50 µl fractions were collected and subjected to SDS-PAGE analysis. Protein bands were visualized by Coomassie staining except for gels containing ASC1<sup>1–80</sup> or ASC1<sup>152–230</sup>, which were imaged by silver staining.
+
+For testing competitive binding of ASC1 and ALKBH3 to ASCC3<sup>HR</sup>, 120 pmol of ASCC3<sup>HR</sup> (or of pre-formed ASCC3<sup>HR</sup>-ASC1 complex) were mixed with 360 pmol each of ASC1 and ALKBH3 (or of ALKBH3) in a volume of 100 µl. After 30 min of incubation on ice, the samples were loaded on a Superdex 200 3.2/300 analytical size exclusion column. 50 µl fractions were collected and subjected to SDS-PAGE analysis. The proteins were visualized by Coomassie staining.
+
+## DNA unwinding assays
+DNA duplex unwinding activity was assessed in fluorescence-based stopped-flow experiments on a SX-20MV spectrometer (Applied Photophysics).<sup>26, 27</sup> The DNA substrate contained a 12-base pair duplex region and a 31-nucleotide 3’-ss overhangs, with an Alexa 488 fluorophore on the short strand and an Atto 540 Q quencher on the complementary strand ([(Atto 540 Q]5’-<b>GGCCGCGAGCCG</b>GAAATTTAATTATAAACCAGACCGTCTCCTC-3’; 5’-<b>CGGCTCGCGGCC</b>-3’[Alexa 488]; duplex region in bold). Reactions were carried out in 40 mM HEPES-NaOH, pH 7.5, 80 mM NaCl, 0.5 mM MgCl<sub>2</sub> at 30°C. 250 nM protein or protein complex were pre-incubated with 50 nM DNA duplex for 5 min. 60 µl of the protein-DNA mixture were rapidly mixed with 60 µl of 4 mM ATP/MgCl<sub>2</sub>, and the excited Alexa 488 fluorescence signal was recorded for 20 min using a 495 nm cutoff filter (KV 495, Schott). For each experiment, at least two individual traces were averaged, baseline-corrected by the fluorescence immediately after addition of ATP and normalized to the baseline-corrected maximum fluorescence. Data for ASCC3<sup>HR,D611A</sup>-based unwinding had been reported previously<sup>20</sup> and are reproduced here to facilitate direct comparison. Data were plotted using GraphPad Prism 6.0 and fitted to a double exponential equation (fraction unwound = <em>A</em><sub>fast</sub>*(1 – exp(–<em>k</em><sub>fast</sub>*<em>t</em>)) + <em>A</em><sub>slow</sub> * (1 – exp(–<em>k</em><sub>slow</sub>*<em>t</em>))); <em>A</em>, total unwinding amplitude; <em>k</em>, unwinding rate constants [s<sup>−1</sup>]; <em>t</em>, time [s]).<sup>25</sup> Amplitude-weighted unwinding rate constants were calculated as <em>k</em><sub>uaw</sub> = (<em>A</em><sub>fast</sub>*<em>k</em><sub>fast</sub><sup>2</sup> + <em>A</em><sub>slow</sub>*<em>k</em><sub>slow</sub><sup>2</sup>) / (<em>A</em><sub>fast</sub>*<em>k</em><sub>fast</sub> + <em>A</em><sub>slow</sub>*<em>k</em><sub>slow</sub>).
+
+## ATPase assays
+Thin layer chromatography (TLC)-based ATPase assays were performed using [α-<sup>32</sup>P]ATP (Hartmann Analytic).<sup>26, 27</sup> To quantify DNA-stimulated ATPase activity, 0.5 µM protein or protein complex were combined with 1 mM of a 43-nt ssDNA (5’-GGCCGCGAGCCGGAAATTTAATTATAAACCAGACCGTCTCCTC-3’). 0.5 µM protein or protein complex or equivalent protein-DNA mixtures were incubated with 1 mM [α-<sup>32</sup>P]ATP in 50 mM HEPES-NaOH, pH 7.5, 80 mM NaCl, 5 mM MgCl<sub>2</sub>, 2 mM DTT at 30°C for up to 60 min. 5 µl of sample were withdrawn at selected time points and reactions were quenched with 5 µl of 100 mM EDTA. 0.8 µl of the samples were spotted on a PEI-cellulose TLC plate and chromatographed with 1 M acetic acid, 0.5 M LiCl, 20% (v/v) ethanol. The corresponding ADP and ATP spots were visualized using a Storm 860 phosphorimager (GMI, USA) and quantified using ImageQuant software (version 5.2; Cytiva). Data were plotted and analyzed using Prism software (Graphpad, version 5), the ATPase activity was calculated as the number of hydrolyzed ATP molecules per protein molecule per minute, by fitting quantified data to the equation <em>V</em> = (<em>A</em><sub>fast</sub>*<em>V</em><sub>fast</sub><sup>2</sup> + <em>A</em><sub>slow</sub>*<em>V</em><sub>slow</sub><sup>2</sup>) / (<em>A</em><sub>fast</sub>*<em>V</em><sub>fast</sub> + <em>A</em><sub>slow</sub>*<em>V</em><sub>slow</sub>); <em>A</em><sub>fast</sub> and <em>A</em><sub>slow</sub>, amplitudes of ATP hydrolyzed in the rapid and slow phase, respectively; <em>V</em><sub>fast</sub> and <em>V</em><sub>slow</sub>, rates of the rapid and slow hydrolysis phases [min<sup>−1</sup>]; <em>V</em>, ATP hydrolyzed as a function of time [min<sup>−1</sup>].
+
+## Fluorescence microscopy
+The sub-cellular localizations of the Flag/His-tagged versions of ASC1 were determined by immuno-fluorescence.<sup>43</sup> 293 cell lines expressing Flag-tagged ASC1 variants were grown on coverslips and fixed using 4% (v/v) paraformaldehyde for 20 min before permeabilization using 0.1% (v/v) Triton-X-100 in PBS for 20 min. Cells were blocked using PBS supplemented with 10% (v/v) fetal bovine serum (FBS) and 0.1% (v/v) Triton-X-100 for 1 h, then treated for 2 h with an FITC-conjugated α-Flag M2 antibody (Sigma Aldrich F4049; 1:200) diluted in PBS containing 10% FBS and 0.1% Triton-X-100. Cells were washed, and coverslips were mounted using mounting media containing DAPI. Cells were imaged using a Nikon Ti2 2-E inverted microscope.
+
+## Immuno-precipitation and western blotting
+293 cells expressing N- or C-terminally Flag/His-tagged versions of full-length or truncated ASC1 or the Flag tag were lysed by sonication in IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.1% (v/v) Triton-X-100, 10% (v/v) glycerol and cOmplete™ protease inhibitors. Lysates were cleared of debris by centrifugation at 20,000 x g for 10 min, then the cleared lysates were incubated with α-Flag M2 magnetic beads (Sigma-Aldrich #M8823) for 2 h. The matrix was washed five times with IP buffer and complexes were eluted using 3xFlag peptide (Sigma Aldrich #SAE0194). Proteins were precipitated using 20% (w/v) trichloroacetic acid (TCA) and separated by SDS-PAGE. Western blotting was performed using antibodies against the Flag tag (Sigma-Aldrich F3165; 1:7500), ASCC1 (Proteintech #12301-1-AP; 1:500), ASCC2 (Proteintech #11529-1-AP; 1:1000) and ASCC3 (Proteintech #17627-1-AP; 1:1000).
+
+For immuno-precipitation of HA-tagged ASC1 variants (ASC1<sup>wt</sup>, ASC1<sup>ΔZnF</sup>, ASC1<sup>L174A−L180A−I190A</sup> or ASC1<sup>C171A−C184A</sup>), the transfected 293T cells were resuspended in ice cold, high salt co-IP buffer (50 mM Tris-HCl, pH 7.9, 300 mM KCl, 10% [v/v] glycerol, 1% [w/v] Triton X-100, 1 mM DTT) supplemented with protease inhibitors. The cells were then lysed by sonication and allowed to rotate at -4°C to complete lysis. Lysates were cleared by centrifugation and diluted to 150 mM KCl using co-IP buffer without KCl. Anti-HA beads (Santa Cruz Biotechnology, sc-7392 AC) were then added to the samples, and after incubation at 4°C for 3.5 h, the beads were centrifuged and washed multiple times with 150 mM KCl co-IP buffer. Bound proteins were eluted with SDS-PAGE loading buffer and boiled before analysis via SDS-PAGE/western blot using antibodies against the HA-tag (Abcam EPR22819-101, 1:4000) and ASCC3 as described previously<sup>13</sup>.
+
+## MMS sensitivity assays
+The wt and ASC1 KO PC-3 cells were plated on a 96-well plate with 3,500 cells per well. Cells were exposed to media containing variable concentrations of MMS for 24 h at 37°C. Then, cells were recovered with fresh culture medium for an additional 48 h at 37°C. Cell viability was measured by using the MTS assay (Promega).
+
+## Cryogenic electron microscopy
+The ASCC3<sup>HR</sup>-ASC1 complex was prepared freshly in buffer 20 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 1 mM DTT, and concentrated to 4.15 mg/ml using a 50k ultra centrifugal filter (Merck). The sample was supplemented with 0.01% (w/v) n-dodecyl β-maltoside promptly before vitrification. 3.8 µl of the sample were applied to glow-discharged holey carbon R1.2/1.3 copper grids (Quantifoil Microtools, Germany) and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher) equilibrated at 10°C and 100% humidity.
+
+Data acquisition was conducted on a FEI Titan Krios G3i TEM operated at 300 kV equipped with a Falcon 3EC detector. Movies were taken for 40.57 s accumulating a total electron flux of ~40 el/Å<sup>2</sup> in counting mode at a calibrated pixel size of 0.832 Å/px distributed over 33 fractions.
+
+## CryoEM data analysis
+All image analysis steps were done with cryoSPARC (version 3.2.2)<sup>44</sup>. Movie alignment was done with patch motion correction generating Fourier-cropped micrographs (pixel size 1.664 Å/px), CTF estimation was conducted by Patch CTF. Class averages of manually selected particle images were used to generate an initial template for reference-based particle picking from 6,022 micrographs. 2,818,857 particle images were extracted with a box size of 160 px and Fourier-cropped to 80 px for initial analysis. Reference-free 2D classification was used to select 1,590,881 particle images for further analysis. <em>Ab initio</em> reconstruction using a small subset of particles was conducted to generate an initial 3D reference for consecutive iterations of 3D heterogeneous refinement. 597,971 particle images were re-extracted with a box of 160 px and subjected to non-uniform refinement followed by CTF refinement. Another heterogeneous refinement round was applied to select 473,863 particle images for re-extraction at full spatial resolution after local motion correction (box size 320 px, 0.832 Å/px). A final heterogeneous refinement run was conducted to select 244,064 particle images for non-uniform refinement and generate the final reconstruction at a global resolution of 3.4 Å, locally extending down to 2.5 Å.
+
+## Model building, refinement and analysis
+AlphaFold-predicted models<sup>24</sup> of ASCC3<sup>HR</sup> and of regions of ASC1 were manually placed in the cryoEM reconstruction and adjusted by rigid body fitting and segmental real-space refinement using Coot (version 0.8.9.1)<sup>45</sup>. The model was refined by iterative rounds of real space refinement in PHENIX (version 1.17.1)<sup>46</sup> and manual adjustment in Coot. Manual adjustments also took advantage of locally refined, focused cryoEM reconstructions. The structural model was evaluated with Molprobity (version 4.5.1)<sup>47</sup>. Interface areas were analyzed via the PISA server (version 1.52)<sup>48</sup>. Structure figures were prepared using ChimeraX (version 1.4)<sup>49</sup> and PyMOL (version 1.8; Schrödinger, LLC).
+
+## DNA-protein cross-linking/mass spectrometry
+UV cross-linking was employed to generated zero length cross-links between protein and bound ssDNA oligos (T<sub>12</sub>, T<sub>24</sub>, T<sub>36</sub>, T<sub>48</sub>). DNA oligos were 5’-end labeled using [γ-<sup>32</sup>P]ATP and T4 polynucleotide kinase using a standard protocol. 10 µl reaction mixtures containing 100 nM (“1” in Fig. <span class="InternalRef" refid="Fig5">5</span> b) or 200 nM (“2” in Fig. <span class="InternalRef" refid="Fig5">5</span> b) protein or protein complex and 4.3 nM radio-labeled DNA probe were incubated in a 72-well microbatch plate (Greiner) in 50 mM HEPES-NaOH, pH 7.5, 80 mM NaCl, 5 mM MgCl<sub>2</sub>, 2 mM DTT on ice for 5 min, then the samples were exposed to 254 nm UV irradiation for 10 min (Ultra-violet cross-linker, Amersham Life Science). Cross-linked samples were separated by SDS-PAGE and visualized by autoradiography using a Storm 860 phosphorimager.
+
+For identifying cross-linked peptides and residues, 6.7 nM unlabeled T<sub>48</sub> ssDNA were cross-linked to 200 nM ASCC3<sup>HR</sup> or ASCC3<sup>HR</sup>-ASC1 in 48 x 10 µl reactions as above and ethanol precipitated. Subsequent analyses were conducted in duplicates. The pellets were dissolved in 50 µl 4 M urea and diluted to 1 M Urea with 50 mM Tris-HCl, pH 7.5. To digest the DNA, 1 µl Universal nuclease (Pierce) and 1 µl Nuclease P1 (New England Biolabs) were added to the samples, followed by incubation at 37°C for 3 h. Protein digestion was performed with 1 µg of trypsin (Promega) overnight at 37°C. The samples were acidified with formic acid (FA; final concentration 0.1% [v/v]), and acetonitrile (ACN) was added to 5% (v/v) final concentration. Non cross-linked nucleotides were depleted by C18 reversed-phase chromatography with Harvard Apparatus MicroSpin columns. Sample was eluted by stepwise application of 50% (v/v) and 80% (v/v) ACN. Cross-linked peptides were enriched over linear peptides by TiO2 self-packed tip columns with 5% (v/v) glycerol as a competitor as described previously<sup>50</sup>. The samples were dried under vacuum and resuspended in 10 to 15 µl of 2% (v/v) ACN, 0.05% (v/v) trifluoroacetic acid. 7 or 8 µl (first or second analysis) were used for LC-MS analysis.
+
+Chromatographic separation was achieved with Dionex Ultimate 3000 UHPLC (Thermo Fischer Scientific) coupled with a C18 column packed in-house (ReproSil-Pur 120 C18-AQ, 1.9/3 µm pore size, 75 µm inner diameter, 30 cm length, Dr. Maisch GmbH). The flow rate was set to 300 nl/min, and a 44 min linear gradient was formed with mobile phase A (0.1% [v/v] FA) and B (80% [v/v] ACN, 0.08% [v/v] FA) from 8% or 10% (first or second analysis) to 45% mobile phase B. Data acquisition of eluting peptides was performed with Orbitrap Exploris 480 (Thermo Fischer Scientific). The resolution for survey scans was set to 120,000, the maximum injection time to 60 ms, the automatic gain control target to 100% or 250% (first or second analysis) and the dynamic exclusion to 9 s. Analytes selected for fragmentation were isolated with a 1.6 m/z window and fragmented with a normalized collision energy of 28. MS/MS spectra were acquired with a resolution of 30,000, a maximum injection time of 120 ms and an automatic gain control target of 100%.
+
+Cross-link data analysis of the resulting raw files was performed with the OpenNuXL node of OpenMS (version 3.0.0)<sup>51</sup>. Default general settings were used and the preset DNA-UV Extended was selected. The sequences of the proteins in the sample were provided as a database. The maximum length of DNA adducts was set to 3 and poly-T was used as sequence. The resulting .idxml files were used for annotation, and spectra were manually validated.
+
+## Data availability
+The cryoEM reconstruction of the ASCC3<sup>HR</sup>-ASC1 complex has been deposited in the Electron Microscopy Data Bank (<span class="ExternalRef"><span class="RefSource">https://www.ebi.ac.uk/pdbe/emdb</span><span address="https://www.ebi.ac.uk/pdbe/emdb" class="RefTarget" targettype="URL"></span></span>) under accession code EMD-15521 (<span class="ExternalRef"><span class="RefSource">https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-15521</span><span address="https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-15521" class="RefTarget" targettype="URL"></span></span>). Structure coordinates have been deposited in the RCSB Protein Data Bank (<span class="ExternalRef"><span class="RefSource">https://www.rcsb.org</span><span address="https://www.rcsb.org" class="RefTarget" targettype="URL"></span></span>) with accession code 8ALZ (<span class="ExternalRef"><span class="RefSource">https://www.rcsb.org/structure/8ALZ)</span><span address="https://www.rcsb.org/structure/8ALZ)" class="RefTarget" targettype="URL"></span></span>).<sup>52</sup> The DNA-protein CLMS data have been deposited in the ProteomeXchange Consortium (<span class="ExternalRef"><span class="RefSource">http://www.proteomexchange.org</span><span address="http://www.proteomexchange.org" class="RefTarget" targettype="URL"></span></span>) via the PRIDE<sup>53</sup> partner repository (<span class="ExternalRef"><span class="RefSource">https://www.ebi.ac.uk/pride/</span><span address="https://www.ebi.ac.uk/pride/" class="RefTarget" targettype="URL"></span></span>) under dataset identifier PXD036106 (<span class="ExternalRef"><span class="RefSource">https://www.ebi.ac.uk/pride/archive/projects/PXD036106</span><span address="https://www.ebi.ac.uk/pride/archive/projects/PXD036106" class="RefTarget" targettype="URL"></span></span>). All other data are contained in the manuscript or the Supplementary Information. Source data are provided with this paper.
+
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+
+# Supplementary Files
+
+- [SupplementaryInformation.docx](https://assets-eu.researchsquare.com/files/rs-2007381/v1/f4ef1a674d4ff0854d564b56.docx)  
+  Supplementary Tables and Figures
+
+- [Sourcedata1gels.pdf](https://assets-eu.researchsquare.com/files/rs-2007381/v1/9035d31f7166528bdb7c479b.pdf)  
+  Supplementary Dataset 1
+
+- [Sourcedata2unwindingATPaseUVCLMMSsurvival.xlsx](https://assets-eu.researchsquare.com/files/rs-2007381/v1/44f45553dc101ab48d51340b.xlsx)  
+  Supplementary Dataset 2
+
+- [Sourcedata3DNAproteinCLMS.xlsx](https://assets-eu.researchsquare.com/files/rs-2007381/v1/eb68bdb50f3751d06cdace80.xlsx)  
+  Supplementary Dataset 3
+
+- [ASCC3ASC1PDBvalidationreport.pdf](https://assets-eu.researchsquare.com/files/rs-2007381/v1/88c59e92bb26d16255558940.pdf)  
+  Supplementary Dataset 4
+
+- [ASCC3ASC1.txt](https://assets-eu.researchsquare.com/files/rs-2007381/v1/2b8bf315656cdfa8b5dc41f9.txt)  
+  Supplementary Dataset 5
+
+- [ASCC3ASC1overall.mrc](https://assets-eu.researchsquare.com/files/rs-2007381/v1/7e36bce74b3ca5aced61fe8f.mrc)  
+  Supplementary Dataset 6
+
+- [ASCC3ASC1focussedonNC.mrc](https://assets-eu.researchsquare.com/files/rs-2007381/v1/c53b710cfcb8263fa9fb69bf.mrc)  
+  Supplementary Dataset 7
+
+- [ASCC3ASC1focussedonCC.mrc](https://assets-eu.researchsquare.com/files/rs-2007381/v1/609dc20432239e132a3d22c4.mrc)  
+  Supplementary Dataset 8