Reference protocol and permeability yardsticks

The MD pipeline in this folder is a deliberate copy of one published protocol, with the numbers it should be read against collected here.

Sources

"Linker-Dependent Folding Rationalizes PROTAC Cell Permeability," J. Med. Chem. 2022 (PMC9574858). The MD paper, and the template for protac_md.py and analyze_conformations.py. Three CRBN/BRD4 PROTACs with different linkers, PEG-like against alkyl.
Atilaw et al., "Solution Conformations Shed Light on PROTAC Cell Permeability," ACS Med. Chem. Lett. 2021, 12, 107-114. NMR plus Monte Carlo conformational search and NAMFIS, not MD, on one VHL PROTAC (ERK5). It is the source of the folded-versus-extended framing and of the Rg cutoff below, nothing else.

Protocol

Setting	Reference choice	Here
Engine / ligand force field	Amber 18, GAFF, geometry pre-optimized in Gaussian	OpenMM 8, OpenFF Sage 2.2 with AM1-BCC charges
Solvent	explicit chloroform, ~3000 molecules, truncated octahedron	explicit chloroform via packmol, plus water as a polar reference
Production	100 ns after equilibration	50-100 ns, `--prod-ns`
Replicas	3	3, independent velocity seeds
Ensemble	300 K, PBC, ~10,000 snapshots per trajectory	300 K / 1 bar NPT, 10 ps per frame
Convergence	RMSD, converged within 5-10 ns	RMSD to the first frame plus replica-mean spread
Analyses	Rg (cpptraj), 3D PSA (PyMOL), intramolecular H-bonds, PMI shape, PCA and K-means into 5 clusters	Rg, 3D PSA (mdtraj SASA over N, O and polar H), H-bonds, PCA and K-means

Chloroform stands in for the membrane interior: its dielectric constant of 4.8 is close to what a molecule sees while crossing a bilayer.

Yardsticks

Rg below 7 A marks the folded, passively permeable regime. This cutoff is from the 2021 NMR paper, on a single VHL PROTAC, and it does not separate the compounds in the 2022 MD paper.

Mean Rg alone does not discriminate. In the 2022 MD paper:

compound	P_passive (nm/s)	dominant Rg	compact local minimum
PROTAC 1, most permeable	30 ± 1.5	just over 8 A	5.6 A
PROTAC 2	11 ± 1.7	just over 7 A	5.5 A
PROTAC 3, least permeable	6 ± 1.4	7-9.5 A, extended	none

Their most permeable compound sits mainly just above 8 A, overlapping their worst. What separates the two is whether a compact minimum near 5.5 A is accessible at all: PROTACs 1 and 2 have one, PROTAC 3 does not. Report the Rg distribution and the compact-state population, which is what rg_basins.py computes, rather than the mean. Absolute Rg is also not strictly transferable across chemical series; theirs is CRBN/BRD4, the runs here are VHL/EGFR.

3D PSA rises as permeability falls: 190-265 A^2 for the two more permeable compounds against 290-330 A^2 for the poorly permeable one. The rough cutoff for low-to-moderate permeability is 200-210 A^2, from the 2021 paper. Absolute values depend on the polar-atom selection; validate_psa.py and psa_method_probe.py pin down where this convention sits relative to theirs.
Permeability correlates inversely with 3D PSA and with intramolecular H-bond count.