Skill: Design Thermostable Mutations

Use When

Use this skill when users need mutation design or triage for proteins/enzymes and care about both:

• improved thermostability/heat tolerance
• preserved catalytic activity/bioactivity

Typical requests:

• "Which positions should I mutate first?"
• "Design a stability-focused mutation library without breaking active site chemistry"
• "Rank these candidate mutations by stability evidence"
• "Build a decision-grade shortlist for wet-lab validation"
• "Predict the stability impact of a specific variant (e.g., a clinical or engineered substitution)"

Decision-Grade Guardrails (Default)

These are enforced by default unless explicitly overridden:

1. Bioactivity constraints must be supplied for design decisions:
   • active-site/catalytic/cofactor/disulfide/blocklist positions
2. Functional/protected positions are blocked from routine mutagenesis.
3. Non-conservative substitutions are rejected in conservative mode.
4. User-facing mutation notation is 1-based and WT residue-validated.

Inputs

• Required:
  • WT sequence (--sequence or --fasta)
• Required for decision-grade design mode:
  • functional constraints (at least one: active/cofactor/disulfide/blocklist)
• Optional:
  • structure (.pdb/.cif/.mmcif)
  • mutation candidates (A123V format)
  • assay context (pH, temperature, ligand/cofactor state)

Workflow

Choose the smallest workflow that still supports decision-grade confidence.

Path A: Fast Heuristic Design (single-script)

Use when structure and external predictors are unavailable.

Script:

• scripts/design_thermostable_mutations.py

Capabilities:

• strict sequence/mutation validation
• protected-residue enforcement (default)
• conservative-substitution enforcement (default)
• heuristic DDG + local aggregation-risk filtering
• ranked candidate output with rejection reasons

Path B: Structure-Aware Consensus (recommended)

Use when structure is available and shortlist quality matters.

0. One-command orchestrator (recommended entrypoint):

• scripts/run_decision_pipeline.py

1. Discover candidate sites with structural context and functional guardrails:

• scripts/discover_candidate_sites.py

2. Generate focused single/multi-site library:

• scripts/generate_mutation_library.py

3. Resolve sequence-to-structure numbering safely:

• scripts/structure_residue_mapper.py

4. Run structure-based scoring (FoldX wrappers):

• scripts/run_foldx_batch.py
• scripts/run_foldx_chunked.py (large libraries)

5. Aggregate multiple metrics with direction-aware consensus:

• scripts/consensus_stability_rank.py

6. Produce decision-ready report using:

• ranked consensus
• support fraction across tools
• functional risk flags
• assumptions and escalation notes

7. Optional AI-assisted route for structure generation/summary:

• scripts/run_colabfold_batch.py

Example:

python3 scripts/run_decision_pipeline.py \
  --sequence-fasta wt.fasta \
  --structure model.pdb \
  --active-site 57,102,195 \
  --cofactor-sites 64,66 \
  --blocklist-sites 1-5 \
  --run-foldx \
  --run-consensus

By default, the pipeline:

• requires functional constraints for decision-grade discovery
• blocks protected sites during library generation
• auto-derives protected sites from active/cofactor/disulfide/blocklist when not explicitly set

Output Contract

Always include:

1. Baseline stability summary
2. Functional-constraint enforcement status
3. Ranked candidates with rationale
4. Rejection summary (rule-level counts)
5. Functional risk flags (active/cofactor/disulfide/interface/conservation)
6. Confidence and escalation section

For consensus workflows, also include:

• per-tool metric table
• consensus score and support fraction
• metric directionality and normalization choices

References

• references/mutation-design-workflow.md
• references/mutation-design-principles.md
• references/stability-prediction-playbook.md
• references/use-case-playbook.md
• references/report-template.md
• references/troubleshooting.md
• references/literature-evidence-2022-2026.md

Evaluation and Bundle Checks (Optional)

• evals.json
• scripts/run_skill_evals.py
• scripts/validate_skill_bundle.py

Install

From repository root:

# Codex (default)
./scripts/install_skill.sh design-thermostable-mutations

# Claude Code
./scripts/install_skill.sh design-thermostable-mutations --tool claude

# Other tools
./scripts/install_skill.sh design-thermostable-mutations --dest ~/.my-tool/skills

Related Skills

• predict-protein-heat-stability: Use this first to assess baseline thermostability of a sequence or compare variant heat stability before launching mutation design. It provides Tm class prediction, composition features, and variant ΔTm analysis without requiring mutation design intent.

Limits

• Heuristic scores are triage evidence, not biochemical truth.
• Predictions must be experimentally validated (Tm/T50/activity).
• If functional constraints are incomplete, activity-preservation confidence drops.
• Path A heuristic DDG model (estimate_ddg_simple) models local hydrophobic core packing; it underestimates surface-exposed positions where Arg/Lys enrichment and salt bridges drive thermostability. Use Path B for surface sites.
• Common failure cases:
  • structure numbering mismatches that are not validated before scoring
  • incomplete active-site/cofactor/disulfide/blocklist constraints
  • overlarge mutation libraries that exceed practical scoring budgets