STRC h09 Phase 4m — Kd_mono soft-floor refinement (pre-registration)
Status: pre-registered. No compute consumed. Defines protocol + thresholds before running so post-hoc selection is impossible.
Goal. Soften the WH2 × F-actin Kd_mono floor used in STRC h09 Phase 4j Avidity Kd Model 2026-04-26 from the Husson 2010 Tβ4 × F-actin = 5–10 mM analog floor down to a comparative-AF3-derived bound. If 4m succeeds, Phase 4j conservative scenario gains a more favorable Kd_mono input → conservative gate (Kd_eff < 50 µM) closes.
Hypothesis
AF3 ipTM is NOT a calibrated Kd predictor (avidity-and-dimers.md §3, Dunbrack ipSAE 2025), but comparative ipTM ratios within a single benchmark family of known-Kd binders can place a soft Kd floor on a related construct of unknown Kd.
Specifically: by running AF3 on the Chereau 2005 ITC-measured WH2 × G-actin family (WAVE2, WASP, WIP, MIM, Tβ4) under matched conditions, we establish a within-family ipTM-vs-Kd relationship. Then run h09 tail91 WH2 × G-actin (same conditions) and project Kd from its ipTM rank.
Caveat baked in: even within a single family, ipTM-Kd correlation is order-of-magnitude at best. Output is a “soft Kd floor” (lower bound on plausible Kd_mono), not a point estimate.
Reference set (Chereau 2005 Fig. 1C, primary lit, all in vault as ~/STRC/literature/actin-kinetics.md)
| WH2 source | Sequence position | Kd × G-actin (ITC) |
|---|---|---|
| WAVE2 | 433–464 (32 aa) | 52 ± 3 nM |
| WIP | 29–60 (32 aa) | 160 ± 10 nM |
| MIM | 724–755 (32 aa) | 230 ± 40 nM |
| WASP | 430–458 (29 aa) | 250 ± 30 nM |
| Tβ4 | 2–44 (43 aa) | 760 ± 120 nM (G-actin only; F-actin floor 5–10 mM cited separately) |
Protocol
AF3 jobs (5 reference + 1 query = 6 total; well under daily 30 quota)
- WAVE2_WH2_433_464 × G-actin trimer (single seed=42)
- WIP_WH2_29_60 × G-actin trimer
- MIM_WH2_724_755 × G-actin trimer
- WASP_WH2_430_458 × G-actin trimer
- Tβ4_WH2_2_44 × G-actin trimer
- h09 tail91 N-term WH2 (RQLVKAIPDNCSKSNVSR, aa 1–18 of construct) × G-actin trimer
All vs G-actin trimer (the Chereau ITC measured G-actin → use G-actin as substrate to keep comparison clean). G-actin trimer = 3 × 375 aa = 1125 aa.
WH2 sequences (need to retrieve from UniProt for reference set):
- WAVE2 (Q9Y6W5) aa 433–464:
[TBD — retrieve before submission] - WIP (Q14155) aa 29–60:
[TBD] - MIM (Q8WUY3) aa 724–755:
[TBD] - WASP (P42768) aa 430–458:
[TBD] - Tβ4 (P62328) aa 2–44:
[TBD] - h09 WH2 (custom):
RQLVKAIPDNCSKSNVSR(already known)
Token budget per job: 32 aa + 1125 aa = 1157 aa (well under 5000).
Analysis
For each reference job, record top-model ipTM. Plot log(Kd_ITC) vs ipTM. Fit linear regression. Apply h09 WH2 ipTM to the line → predicted log(Kd) for h09 WH2 × G-actin.
Then propagate: h09 Kd × F-actin = h09 Kd × G-actin × scaling_factor. Scaling factor from Tβ4 family: Tβ4 × G-actin = 760 nM, Tβ4 × F-actin = 5–10 mM → ratio ≈ 6,500–13,000×. Apply ratio to predicted h09 Kd × G-actin.
Pass criteria (pre-registered)
- R² of family fit ≥ 0.5. If reference family fit is too noisy, abandon the comparison; soft-floor cannot be established.
- h09 ipTM lies within reference range. Extrapolation outside (0.4–0.7) is unreliable; if h09 ipTM < 0.4 or > 0.75, abandon.
- Predicted h09 Kd × G-actin × F-actin scaling < 5 mM. This becomes the soft floor that replaces Husson Tβ4 5–10 mM in Phase 4j.
- If predicted h09 Kd_mono < 1 mM, conservative Phase 4j scenario closes: Kd_eff = 1e-3 × (1e-3/0.05)^3 = 8 µM (PASS gate 50 µM).
What this does NOT prove
- Does NOT measure absolute Kd. Only places a soft floor for Phase 4j input.
- Does NOT validate AF3 ipTM as Kd predictor in general (it isn’t). Within-family comparative ratios are the only legitimate use.
- Does NOT close the wet-lab gate (which is off-limits per project rules); it tightens the computational substitute.
Cost
6 AF3 jobs (1 quota cycle, ~25 min wall-time). No M5 compute. Pure analytic afterward.
Status
Pre-registered 2026-04-26 16:??. Not submitted. Submit when AFS quota refreshes (~midnight UTC after retrieving WH2 reference sequences from UniProt).
Connections
[part-of]STRC Synthetic Peptide Hydrogel HTC (h09 hub)[informs]STRC h09 Phase 4j Avidity Kd Model 2026-04-26 (Kd_mono input)[uses]2026-04-23-chereau-wh2-actin-pnas (Fig. 1C reference Kd set)[uses]~/STRC/literature/actin-kinetics.md(Tβ4 G→F-actin scaling factor)[caveat]~/STRC/literature/avidity-and-dimers.md§3 (AF3 ipTM is NOT a Kd predictor; comparative-only)