double_diff/README.md

Double Diff Screening Pipeline

Overview

This pipeline implements a dual-dimension differentiation screening strategy to identify prophage-encoded proteins that exhibit significant divergence from known Anti-CRISPR (Acr) proteins at both the sequence and structural levels. The objective is to discover highly confident novel Acr candidates that likely utilize unprecedented, independent mechanistic modalities.

Screening Strategy

The pipeline employs an integrated sequence-structure filtering framework. By systematically evaluating and excluding proteins that demonstrate significant sequence homology or structural conservation with experimentally validated Acr proteins (2025-verified dataset), we isolate "double-divergent" candidates.

Input Data

Data Type	Source Description
Query Sequences	Prophage-encoded proteins extracted from self-targeting bacterial genomes
Reference Sequence Database	2025-verified experimentally validated Acr protein sequences
Reference Structure Database	2025-verified experimentally validated Acr protein 3D structures

Screening Workflow

Stage 1: Sequence Divergence Screening (BLASTP-based)

Candidate prophage proteins are subjected to homology screening against the 2025-verified Acr sequence reference database to isolate targets with independent evolutionary trajectories:

• Alignment Tool: BLASTP
• Threshold Parameter: E-value ≤ 1E-5
• Evaluation Metrics: Query Coverage (qcovs), Percent Identity (pident)
• Exclusion Criteria: qcovs ≥ 30 AND pident ≥ 30
• Retention Criteria: No significant BLASTP hits OR (qcovs < 30 OR pident < 30)

Note: Proteins retained at this stage include those that yield absolutely no hits against the database, as well as those with weak alignments failing the exclusion criteria, ensuring true sequence novelty.

Associated Script: gain_rm_seq_similar_seqs.py

Stage 2: Structural Divergence Screening (Foldseek-based)

Sequence-divergent candidates from Stage 1 undergo three-dimensional structural comparison to evaluate fold novelty:

• Alignment Tool: Foldseek
• Evaluation Metrics:
  • inhibition_type_probability: Probability of structural fold similarity
  • alntmscore: Structural alignment TM-score
• Screening Criteria: inhibition_type_probability = NA AND alntmscore = NA
• Length Filter: Sequence length ≥ 50 amino acid residues (seq_len ≥ 50)

Note: Foldseek does not output records for pairs without significant structural homology. Consequently, candidates lacking any structural match to the reference database are merged into the final dataset with NA (Not Available) for all structure-related metrics.

Stage 3: Result Integration and Output

Final screening results are compiled in double_diff_foldseek.csv with the following fields:

Field	Description
query	Candidate protein unique identifier
target	Best structural match reference protein (null if no valid match)
inhibition_type_probability	Structural fold similarity probability (0 indicates no structural match)
alntmscore	Structural alignment TM-score (0 indicates no significant alignment)
lddt	Local Distance Difference Test score
Acr_Type	Reference protein inhibition type classification (null if no match)
seq	Amino acid sequence
seq_len	Sequence length in amino acid residues (filtered for ≥ 50)
blastp_pident	BLASTP percent identity (0 for no hits/excluded)
blastp_qcovs	BLASTP query coverage (0 for no hits/excluded)

Screening Logic Diagram

┌─────────────────────────────────────────────────────────────────┐
│              Prophage Protein Candidate Dataset                 │
└──────────────────────────┬──────────────────────────────────────┘
                           │
                           ▼
┌─────────────────────────────────────────────────────────────────┐
│  Stage 1: Sequence Homology Screening (BLASTP)                  │
│  - E-value ≤ 1E-5                                               │
│  - Exclude: qcovs ≥ 30 AND pident ≥ 30                          │
│  - Retain: No hits OR below exclusion thresholds                │
└──────────────────────────┬──────────────────────────────────────┘
                           │
              ┌────────────┴────────────┐
              ▼                         ▼
       ┌──────────────┐          ┌──────────────┐
       │  Sequence-   │          │  Sequence-   │  ← Exclude
       │  Divergent   │          │  Similar     │
       │  (Retain)    │          │  (Exclude)   │
       └──────┬───────┘          └──────────────┘
              │
              ▼
┌─────────────────────────────────────────────────────────────────┐
│  Stage 2: Structural Similarity Screening (Foldseek)            │
│  - alntmscore = NA (No Foldseek hit)                            │
│  - inhibition_type_probability = NA (No Foldseek hit)           │
│  - seq_len >= 50                                                │
└──────────────────────────┬──────────────────────────────────────┘
                           │
              ┌────────────┴────────────┐
              ▼                         ▼
       ┌──────────────┐          ┌──────────────┐
       │  Structure-  │          │  Structure-  │  ← Exclude
       │  Divergent   │          │  Similar     │
       │  ★ Final     │          │  (Exclude)   │
       │  Candidates  │          │              │
       └──────────────┘          └──────────────┘