| # Double Diff Screening Pipeline |
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| ## Overview |
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| 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. |
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| ## Screening Strategy |
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| 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. |
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| ## Input Data |
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| | 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 | |
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| ## Screening Workflow |
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| ### Stage 1: Sequence Divergence Screening (BLASTP-based) |
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| Candidate prophage proteins are subjected to homology screening against the 2025-verified Acr sequence reference database to isolate targets with independent evolutionary trajectories: |
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| - **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) |
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| *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.* |
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| **Associated Script**: `gain_rm_seq_similar_seqs.py` |
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| ### Stage 2: Structural Divergence Screening (Foldseek-based) |
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| Sequence-divergent candidates from Stage 1 undergo three-dimensional structural comparison to evaluate fold novelty: |
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| - **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) |
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| *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.* |
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| ### Stage 3: Result Integration and Output |
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| Final screening results are compiled in `double_diff_foldseek.csv` with the following fields: |
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| | 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) | |
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| ## Screening Logic Diagram |
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| ```text |
| ┌─────────────────────────────────────────────────────────────────┐ |
| │ 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 │ │ │ |
| └──────────────┘ └──────────────┘ |
