STRC h01 Phase 9 — Orthogonal Cross-Checks Plan
Plan note. Five tracks of independent in-silico validation prepared as runnable skeletons. No production runs yet — this document defines the menu, the verifiable pass criteria, and the smoke-test rules so the production runs can fire tomorrow/day-after.
Why this phase exists
The h01 differential (mutant E1659A vs WT for chaperone-pocket binding) currently rests on three pillars:
- Vina / Boltz-2 docking consensus (Phase 5e, 5q) — known artifacts: pose-search bias, force-field charge approximations, scoring-function calibration error.
- tauRAMD residence times (Phase 5m, 5q) — known artifacts: forced-pull non-equilibrium, force constant choice, mass scaling.
- APBS continuum electrostatics (Phase 5j, 5k, 8g-v2) — known artifacts: PB box geometry, dielectric assumption, no explicit waters/ions in pocket.
Each pillar can be wrong in a way that conspires with the other two (shared force-field family, shared docking-derived starting poses). Reviewers — and an honest internal audit — need cross-checks that use different physics (FEP, PMF, QM) or different model families (independent AI co-folders) to confirm the differential survives.
Tracks (skeletons exist, awaiting production runs)
| # | Method | Tests | Skeleton |
|---|---|---|---|
| 9a | FEP/TI alchemical ΔΔG | absolute/relative binding free energy via λ-mutation E→A | STRC h01 Phase 9a FEP-TI E1659A Alchemical 2026-04-27 SKELETON |
| 9b | Holo MD + MM-PBSA | long-trajectory complex stability + isolated electrostatic ΔG | STRC h01 Phase 9b Holo MD MM-PBSA 2026-04-27 SKELETON |
| 9c | Umbrella sampling PMF | equilibrium dissociation barrier (replaces forced-pull tauRAMD) | STRC h01 Phase 9c Umbrella Sampling PMF 2026-04-27 SKELETON |
| 9d | QM/MM single-point + opt | quantum treatment of charged-residue pocket; polarization/charge-transfer | STRC h01 Phase 9d QM-MM Pocket 2026-04-27 SKELETON |
| 9e | Blind neural docking | independent AI co-fold (AF3 / DiffDock / Boltz-2 / Uni-Mol) | STRC h01 Phase 9e Blind Neural Docking AF3 DiffDock 2026-04-27 SKELETON |
Pass criteria for the phase as a whole
- PRIMARY: ≥3 of 5 tracks reproduce the sign of the existing differential (mutant binds better than WT).
- SECONDARY: at least one of {9a, 9c} produces a quantitative ΔΔG estimate within 2 kcal/mol of the MM-PBSA value (9b).
- TERTIARY: 9e blind co-fold places lead ligand in the K1141 pocket for ≥2 of 4 AI models without seeded coordinates.
- FAIL state: ≥3 tracks show no differential or opposite sign → freeze h01 wet-lab planning, return to mechanism re-derivation.
Smoke-test rule (applies to every track)
Each skeleton specifies a 1-day smoke variant that runs on existing AF3 / Phase 5d MD / Phase 5q docked assets — no re-equilibration. Smoke output is a binary go/no-go on the production protocol, not a scientific result. Production fires only after smoke passes.
Candidate extras — menu of further cross-checks (not skeletoned yet)
If the 5 main tracks come back ambiguous or a reviewer asks for more, these are the next-tier options ordered by orthogonality / ease:
| Extra | What it adds | Cost |
|---|---|---|
| Metadynamics / well-tempered MetaD | faster PMF than umbrella; orthogonal CV biasing | medium |
| GaMD (Gaussian-Accelerated MD) | enhanced sampling without CV definition | low-medium |
| WaterMap / GIST / 3D-RISM | water-displacement thermodynamics in pocket | low |
| Markov State Models from existing MD | kinetic decomposition orthogonal to single-trajectory tauRAMD | low (post-process) |
| LIE (Linear Interaction Energy) | cheap end-state alternative to FEP | very low |
| MM-GBSA per-residue decomposition | residue-specific energy contribution to differential | very low (post-process) |
| FTMap / SILCS / MixMD solvent probe | orthogonal pocket druggability map | low |
| DCCM / network analysis on MD | allosteric signal from E1659 to K1141 pocket | low (post-process) |
| Force-field cross (AMBER ↔ CHARMM ↔ OpenFF) | robustness to FF family | medium |
| Water-model cross (TIP3P ↔ OPC ↔ TIP4P-Ew) | robustness to water parameterization | medium |
| Charge-isosteric mutational panel (E1659D / E1659K / E1659G) | does mechanism follow charge? | medium |
| ESMFold / OmegaFold structural prior comparison | orthogonal structure to AF3 starting models | low |
These are documented here for future scoping, not yet skeletoned as phase notes. Promote to a Phase 9f-9q skeleton only when the corresponding decision becomes load-bearing.
Tooling readiness — pre-flight matrix (to fill in before launching)
| Track | Software | License? | Hardware? | Existing assets reusable? | Smoke wall-time |
|---|---|---|---|---|---|
| 9a | GROMACS-FEP / AMBER pmemd / pmx | TBD | GPU (M5 Max MPS or remote A100) | Phase 5d holo trajectory | TBD — fill before run |
| 9b | GROMACS + gmx_MMPBSA / AmberTools | installed locally; input-blocked | GPU for production, local CPU OK for setup | Phase 5q E1659A OpenMM assets only; matched WT holo missing | smoke blocked until matched WT/MUT topologies exist |
| 9c | GROMACS pull + gmx wham | TBD | GPU | Phase 5d starting frame | TBD |
| 9d | ORCA + AMBER / CP2K / ChemShell | TBD | CPU-heavy (cluster?) | Phase 5e shortlisted poses | TBD |
| 9e | AF3 server + DiffDock + Boltz-2 + Uni-Mol | already integrated for AF3/Boltz-2 | local + AF3 server | Phase 5q shortlist + ligand SMILES | TBD |
Each row gets filled inside its respective skeleton before that track fires.
Status
- 2026-04-27 00:30 GMT+8 — Phase 9 menu opened; 5 skeleton phase notes created; tooling matrix unfilled. No production runs yet.
- 2026-04-27 11:29 GMT+8 — Phase 9b-lite residue-field smoke delivered from existing matched WT/MUT MD snapshots: broad shells support E1659A>WT sign (18 A delta +0.181 q/A, p=1.54e-10), with 9 A inner-shell caveat. This is supportive smoke, not one of the 5 production track verdicts. → STRC h01 Phase 9b-lite Residue Field Decomposition 2026-04-27
- 2026-04-27 11:45 GMT+8 — Phase 9x v5.3 alpha sensitivity delivered:
adamantyl_acylsulfonamide_SO2Me_-Clstays rank-1 for alpha 0.5-2.0 across Boltz-2, Vina, tauRAMD, and ADMET rank axes. Supportive lead-committee robustness; not a production track verdict. → STRC h01 Phase 9x v5.3 Lead Committee Alpha Sensitivity 2026-04-27 - 2026-04-27 11:54 GMT+8 — Phase 9b MM-PBSA tooling preflight delivered: GROMACS / gmx_MMPBSA / AmberTools callable, but WT-vs-E1659A MM-PBSA is input-blocked. No native GROMACS/Amber h01 topologies exist; Phase 5q has only E1659A OpenMM holo assets; post-hoc OpenMM → Amber conversion is invalid due to missing bond types. → STRC h01 Phase 9b MM-PBSA Tooling Preflight 2026-04-27
Ranking delta
- h01: no change. This is a planning + cross-check infrastructure phase. Tier/mech/deliv/misha_fit/next_step do not move until at least one of 9a-9e returns a verdict. Recording this so the no-change is auditable.
Connections
[parent]h01 hub[see-also]STRC h01 Phase 5m Production tauRAMD Ranking 2026-04-26 — pillar #2 this phase stress-tests[see-also]STRC h01 Phase 5q v5.3 Acyl Sulfonamide Boltz-2 + Vina Consensus 2026-04-26 — pillar #1[see-also]STRC h01 Phase 5j APBS WT vs Mutant Pocket Electrostatics 2026-04-24 — pillar #3[see-also]STRC h01 State Audit 2026-04-26[child]STRC h01 Phase 9a FEP-TI E1659A Alchemical 2026-04-27 SKELETON[child]STRC h01 Phase 9b Holo MD MM-PBSA 2026-04-27 SKELETON[child]STRC h01 Phase 9c Umbrella Sampling PMF 2026-04-27 SKELETON[child]STRC h01 Phase 9d QM-MM Pocket 2026-04-27 SKELETON[child]STRC h01 Phase 9e Blind Neural Docking AF3 DiffDock 2026-04-27 SKELETON[about]Misha