Methodology Notes

The following methodology notes are documented for transparency. Full detail is in DESIGN_PROVENANCE.md and in the methodology_notes field of validation/all_predictions_summary.json.

Note 1 - Rosetta ΔΔG for D8PEA4 / D7BKC8 (cross-protein invalid)

Note 4 in DESIGN_PROVENANCE.md.

D8PEA4 and D7BKC8 are natural IS110 orthologs included in Strategy D. Their Rosetta cartesian_ddg values were computed using IS621 as the reference structure - this produces cross-protein absolute energies, not thermodynamic ΔΔG values. Values set to None; ddg_method = rosetta_cross_protein_INVALID.

Note 2 - Rosetta stability gate universally non-functional

Note 5 in DESIGN_PROVENANCE.md.

ALL 47 Rosetta cartesian_ddg values (Strategy A: 15, C: 2, D: 30) are cross-structure absolute energies (range -31,838 to -41,308 kcal/mol), not point-mutation ΔΔG values. The gate was not applied to any design. Structural quality is instead proxied by ESMFold pLDDT (global mean > 70; active-site > 70 for all triaged designs).

Disclosure (mandatory): “The Rosetta stability gate was non-functional for all designs due to cross-protein absolute energy computation. No designs were excluded on stability grounds. ESMFold pLDDT is used as a structural quality proxy.”

Note 3 - MHCflurry version shift

Strategy D S_Immuno scores individually recomputed with MHCflurry 2.2.1. Strategy B uses the IS621 conservative baseline (0.7594) rather than per-design computation. The calibrated IS621 lockpoint (0.9255) is reported as a secondary analysis alongside the verbatim 0.929.

Note 4 - P3 reference corrected

The earlier placeholder WT_IS621_S_IMMUNO = 0.250 is incorrect. The correct value is 0.7594. Delta for IS621_deimmunized_v2 = +0.1183 >= 0.10 -> PASS.

Note 5 - P5 diversity enforcement

Rank-5 was diversity-enforced: A_007 (pen_score = 0.9209, Strategy A) replaces the natural rank-5 D023 (pen_score = 0.9319, Strategy D) to satisfy the >= 3 strategy criterion. The top-5 is not purely rank-ordered.

Note 6 - Gate 8 ATLAS novelty not evaluated for Strategy B

The GENOME-ATLAS DuckDB embedding-distance gate was not evaluated in this run (database access pending). Strategy B designs were pre-filtered for genus diversity (700 distinct genera) at the sourcing step, providing de facto novelty evidence. Disclosed in validation/P4_orthologs_result.json.