Design on What Was Actually Done

 Quick Analysis Plan Copied

 Long Analysis Plan

1. Evidence anchor: what the ClCRY4 HDX-MS baseline establishes

The primary ClCRY4 study (ClCRY4 pump-probe HDX-MS primary study) provides the critical operational parameters your magnetic-field experiment must reuse: 5 µM ClCRY4, 90% D2O, pH 7.6, 5-min HDX probe, pepsin digestion, and 450 nm/250 lux pump times from 10 s to 20 min. Lowering temperature to 10 °C and 5 °C stabilizes FADH• with minimal decay over 20 min — this is the single most important design choice, because it extends the lifetime of the radical-pair-associated state during which a magnetic field could plausibly alter spin chemistry (Temperature-dependent FADH• stability in ClCRY4). The seven-peptide light-protection signature gives you a known positive readout; peptide 232-243's bimodal, light-dependent population shift toward the protected conformer (quantified by double-binomial fitting in HX-Express v3) is an unusually clean, ratio-based metric that is less sensitive to absolute back-exchange errors than centroid Δ%D (Bimodal phosphate-binding-loop population shift under light).

2. Experimental matrix: embedding a magnetic field into the validated workflow

Field conditions (no supplied record reports magnetic-field HDX data — these magnitudes are design proposals, not sourced effect sizes): (a) shielded null (~µT, mu-metal enclosure); (b) Earth-strength static (~50 µT); (c) intensified static (~100–200 µT); (d) a field applied only during the pump; (e) a field applied only during the 5-min HDX probe. Applying the field during the pump tests whether radical-pair spin dynamics alters the conformational state formed; applying it during the probe tests whether field effects contaminate exchange itself (a critical artifact control). Controls: E505D, which abolishes most light-induced protection while retaining a WT-like response in peptide 482-495 (E505D clamp mutant as mechanistic negative control), serves as the mechanistic null: any field-dependent Δ%D appearing in E505D indicates a field artifact on exchange or MS, not radical-pair biology. Dark-state, no-light, and zero-field runs bracket every condition. Replication: the source study used only 2 biological replicates; for a small, hypothesized field effect, plan ≥5 biological replicates per condition and interleave field-on/field-off runs within the same day to absorb instrument drift, following interlaboratory HDX-MS variance lessons from the NISTmAb benchmark (~78,900 Fab centroids across 15 labs, 3.6–25.4 °C) (NISTmAb interlaboratory HDX-MS reproducibility dataset).

3. Computational/analysis plan

(i) Uptake processing: centroid fitting with back-exchange correction via fully deuterated controls, enforcing EX2 behavior checks as in the NIST protocol. (ii) Primary endpoint: Δ%D per peptide as a function of pump time × field condition, modeled with a mixed-effects design (peptide, pump time, field strength, field phase as fixed effects; biological replicate as random effect) using statsmodels/numpy. (iii) Secondary endpoint: fraction of the protected conformer in peptide 232-243 via double-binomial fitting (port HX-Express v3 logic to Python scipy.optimize). (iv) Ensemble validation: map significant Δ%D onto PDB 6PU0 / AlphaFold model and test candidate conformational ensembles using the ValDX framework — spatial/random data splits and Work-Done metrics to distinguish real field-induced protection shifts from overfitting, since training error alone cannot discriminate ensemble quality (ValDX ensemble-validation framework for HDX-MS). (v) Power before data: Monte-Carlo simulate Δ%D detection limits from the NIST-derived replicate variance to define the minimum detectable field effect — if the minimum detectable effect exceeds any plausible radical-pair modulation, the experiment cannot succeed and should be redesigned (longer pump accumulation, 5 °C, higher field gradients).

4. Known gaps and falsification

The supplied records contain no direct magnetic-field measurements on ClCRY4 or any HDX-MS system — the claimed magnitude of radical-pair magnetic sensitivity is the largest unknown, and the 10 s temporal floor of pump-probe HDX cannot resolve sub-second spin dynamics, only their downstream conformational consequences. The design is falsified if (a) field-on Δ%D shifts occur identically in dark runs or E505D (artifact), or (b) no field condition alters the seven-peptide signature at powered resolution, which would constrain (though not falsify) the radical-pair conformational-signaling hypothesis. HDX-MS interpretation pitfalls — peptide-level resolution limits, back-exchange, and allosteric misattribution — apply throughout (HDX-MS drug discovery methodology review).Generate Full HDX-MS Field ProtocolCreate Field-Sensitivity HypothesisPlan Statistical AnalysisRun AI Scientist AnalysisMake MovieMake Poster Evolve Bioinformatics/Coding/Analysis Plan

Figure note: values are as reported in the source record — E505D retains ~40% FADox after 20 min illumination and a ~50–60% FADH• plateau; WT shows near-complete conversion (plotted midpoint with nominal uncertainty for the reported range). Field-dependent targets are the seven light-protected peptides, not these photoreduction endpoints.