Tooluniverse Residue Functional Mechanism Interpretation

Residue functional mechanism interpretation

The core user question: "Why are these residues functionally critical?"

Answering needs more than one source: a residue in the ligand pocket means something different from one in a catalytic triad, an interface, the hydrophobic core, or a PTM sequon. This skill synthesizes evidence from multiple TU tools to call a mechanism for each residue (or cluster of adjacent residues).

The residues can come from any source — this skill is agnostic to where they came from:

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When to use this skill

• You have a list of residues (from anywhere) and need to explain why they

matter biologically

• You're writing the methods/discussion section of a paper and need

mechanistic claims per residue or cluster

• You're comparing residue sets across orthologs and need a per-residue

category to align by

Not for:

• Validating a predictor against DMS — use

tooluniverse-variant-predictor-dms-validation

• Single-variant SAE feature decomposition when you want to see which

ESMC features changed — use tooluniverse-protein-sae-variant-interpretation

• Single-variant LoF mechanism synthesis for one variant in isolation

— use tooluniverse-protein-lof-mechanism

The single-variant skills focus on one mutation's signature; this skill focuses on which residues matter and why.

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Required inputs (choose one entry path)

Path A — Residues supplied directly (covers ClinVar / literature / custom positions):

Path B — Detect hotspots from a DMS map (original use case):

In Path B the skill runs Step 1 to detect hotspots; in Path A it skips Step 1 entirely and goes straight to Step 2 (gather evidence).

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Workflow

Step 0 (MANDATORY if user names specific positions): Premise check

If the user says "explain why residue/cluster X is a hotspot", do NOT take that as a given. Verify it's actually a hotspot in THIS DMS first — users import biological knowledge from other contexts that may not match what the specific assay measured.

# Per-position disruption magnitude (same formula as Step 1)
if disruptive_tail == "top":
    dms_per_pos = np.nanmax(dms_matrix, axis=0)
elif disruptive_tail == "bottom":
    dms_per_pos = -np.nanmin(dms_matrix, axis=0)

# Where do the user-named positions actually rank?
ranks = (-dms_per_pos).argsort().argsort()  # 0 = highest
for user_pos in user_named_positions:
    rank = int(ranks[pos_index[user_pos]])
    pct = 100 * (1 - rank / len(dms_per_pos))
    print(f"  pos {user_pos}: rank {rank+1}/{len(dms_per_pos)}, "
          f"top {pct:.0f}% by max effect")

Decision rule:

• If named position is in top 25% by max effect → premise confirmed, proceed.
• If named position is in top 50% but not 25% → premise weakly supported;

proceed but note the rank in your report.

• If named position is below top 50% → REPORT THIS MISMATCH TRANSPARENTLY

at the top of your answer before continuing with the mechanism analysis.

Concrete example: the user asks "why is KRAS G12/G13 a folding hotspot in this DMS?" The data say G12 ranks 105/187 (top 56%) and G13 ranks 124/187 (top 66%) by max ΔΔG — they are NOT folding hotspots in this AbundancePCA assay (even though they ARE famous oncogenic positions). The skill's job is to surface that contradiction up front, then proceed with a mechanism analysis for those residues (their oncogenic effect is via GTPase abolishment, not fold disruption — and that's a genuinely useful answer to the user's actual scientific question, just not the one they literally asked).

Step 1 (Path B only): Detect hotspots from the DMS matrix

Skip this step entirely if the user already provided positions (Path A). In Path A the residue list IS the input; jump straight to clustering at the bottom of this section.

import numpy as np

if user_provided_positions:
    # Path A — residues from any source (ClinVar / literature / custom)
    positions_to_analyze = sorted(set(user_provided_positions))
else:
    # Path B — detect from DMS matrix
    if disruptive_tail == "top":
        dms_per_pos = np.nanmax(dms_matrix, axis=0)   # max destabilization at any allele
    elif disruptive_tail == "bottom":
        dms_per_pos = -np.nanmin(dms_matrix, axis=0)  # flip for low-is-bad assays

    K = 20
    positions_to_analyze = sorted(np.argsort(-dms_per_pos)[:K].tolist())

# Chain adjacent positions (gap ≤ 2) into clusters — shared by both paths
clusters = []
current = [positions_to_analyze[0]]
for p in positions_to_analyze[1:]:
    if p - current[-1] <= 2:
        current.append(p)
    else:
        clusters.append(current)
        current = [p]
clusters.append(current)
print(f"{len(clusters)} cluster(s): {clusters}")

A "cluster" of one is allowed — it just gets less statistical power in the permutation test, but multi-evidence interpretation still works.

Path A workflow contracts (what's available vs not):

Step 2: Gather structural evidence per cluster

Annotate the protein structure once, then read fields for each cluster's positions:

# One-shot structural annotation (cached for the rest of the skill)
struct = Structure_annotate_per_residue(
    pdb_id="6VJJ",                 # pick a structure with the relevant complex
    target_chain="A",
    partner_chains=["B"],          # if there's a binding partner
    ligand_resnames=["GNP", "MG"], # if there's a relevant ligand
    distance_cutoff=5.0,
    include_secondary_structure=True,
)
by_pos = {r["position"]: r for r in struct["data"]["annotations"]}

for cluster in clusters:
    structural_summary = {
        "interface_count": sum(1 for p in cluster if by_pos.get(p, {}).get("region") in ("interface", "both")),
        "ligand_pocket_count": sum(1 for p in cluster if by_pos.get(p, {}).get("region") in ("ligand", "both")),
        "core_count": sum(1 for p in cluster if by_pos.get(p, {}).get("is_core")),
        "ss_elements": [by_pos.get(p, {}).get("ss_element") for p in cluster],
    }

Step 3: Gather UniProt feature evidence per cluster

# UniProt features per position (active sites, binding sites, PTM sites, etc.)
up = UniProt_get_function_by_accession(accession="P01116")
# Parse up["features"] into a per-position lookup
features_by_pos = {}
for feat in up.get("features", []):
    start = int(feat.get("begin", feat.get("position", -1)))
    end = int(feat.get("end", start))
    for p in range(start, end + 1):
        features_by_pos.setdefault(p, []).append({
            "type": feat.get("type"),
            "description": feat.get("description", ""),
        })

for cluster in clusters:
    uniprot_summary = {
        "active_site": [p for p in cluster if any(f["type"] == "Active site" for f in features_by_pos.get(p, []))],
        "binding_site": [p for p in cluster if any(f["type"] == "Binding site" for f in features_by_pos.get(p, []))],
        "modified_residue": [p for p in cluster if any(f["type"] == "Modified residue" for f in features_by_pos.get(p, []))],
        "disulfide_bond": [p for p in cluster if any(f["type"] == "Disulfide bond" for f in features_by_pos.get(p, []))],
        "domain": [f.get("description") for p in cluster for f in features_by_pos.get(p, []) if f.get("type") == "Domain"],
    }

Step 4 (optional): Per-cluster SAE feature ranking

Two paths depending on whether you already have a full DMS SAE tensor:

Path A — no precomputed tensor (most cases): use the region tool directly. If the cluster is a contiguous range (or you can pad to one), ESM_get_region_sae_features aggregates SAE features over the range in a single Forge call:

region = ESM_get_region_sae_features(
    sequence=ref_sequence,
    start_position=min(cluster_positions),
    end_position=max(cluster_positions),
    top_k_features=5,
)
top_features = [f["feature_id"] for f in region["data"]["top_features"]]

This is the right default — 1 Forge call vs 20 × cluster-size for the DMS-tensor path. For non-contiguous clusters, run once per contiguous sub-range and union the top-K.

Path B — you already have a precomputed SAE tensor from a DMS sweep (e.g. the variant-predictor-dms-validation pipeline left one on disk): compute drops directly without re-calling Forge.

def cluster_sae_features(sae_tensor, wt_vec, cluster_positions, top_n=5):
    """Returns top SAE features by mean drop at cluster, ready for labeling."""
    drops = np.maximum(0.0, wt_vec[None, :, :] - sae_tensor)
    max_drop_per_pos = np.nanmax(drops, axis=0)  # (n_pos, 16384)
    cluster_mean = max_drop_per_pos[cluster_positions].mean(axis=0)
    return np.argsort(-cluster_mean)[:top_n].tolist()

top_features = cluster_sae_features(sae_tensor, wt_vec, cluster_cols, top_n=5)

Label each top feature via the SAE feature labeler:

for f in top_features:
    label = ESM_describe_sae_feature(feature_id=int(f), n_proteins=5)
    print(f"  feature {f}: {label['data'].get('category')} (conf {label['data'].get('confidence')})")

The first label call for each feature is slow (~30s, ~10 Forge credits as the labeler runs SAE on a 10-protein panel); subsequent calls hit cache.

Step 4-alt: permutation-test the SAE features (optional, more rigorous)

def permutation_pvalues(cluster_positions, max_drop, n_perm=10000, rng=None):
    """Per-feature: is the cluster's mean drop > random equally-sized set?"""
    rng = rng or np.random.default_rng(0)
    n_positions = max_drop.shape[0]
    cluster_size = len(cluster_positions)
    observed = max_drop[cluster_positions].mean(axis=0)
    null_geq = np.zeros(max_drop.shape[1], dtype=np.int32)
    for _ in range(n_perm):
        idx = rng.choice(n_positions, size=cluster_size, replace=False)
        null_geq += (max_drop[idx].mean(axis=0) >= observed).astype(np.int32)
    return (null_geq + 1) / (n_perm + 1)

from statsmodels.stats.multitest import multipletests
p_raw = permutation_pvalues(np.array(cluster_cols), max_drop_per_pos)
_, p_adj, _, _ = multipletests(p_raw, method="fdr_bh")
significant_features = np.where(p_adj < 0.05)[0].tolist()

Use the mean of max_drop, not the max — under this null a maximum-based statistic returns almost no significant features. Single-position clusters return no significant features (no statistical power) — fall back to Step 4 descriptive ranking.

Step 5: Synthesize the mechanism call

For each cluster, combine the 3 (or 4 with SAE) evidence streams:

def call_mechanism(structural, uniprot, sae_labels=None):
    """Return one of: catalytic | ligand-binding | interface |
    structural-core | PTM | regulatory | mixed | unknown."""

    # Direct UniProt evidence wins
    if uniprot["active_site"]:
        return "catalytic"
    if uniprot["binding_site"]:
        return "ligand-binding"
    if uniprot["modified_residue"] and len(uniprot["modified_residue"]) >= len(cluster) // 2:
        return "PTM"

    # Structural evidence
    if structural["ligand_pocket_count"] >= len(cluster) // 2:
        return "ligand-binding"
    if structural["interface_count"] >= len(cluster) // 2:
        return "interface"
    if structural["core_count"] >= len(cluster) // 2:
        return "structural-core"

    # SAE evidence as tiebreaker
    if sae_labels:
        from collections import Counter
        cat_counts = Counter(l for l in sae_labels if l)
        if cat_counts:
            top_cat, n = cat_counts.most_common(1)[0]
            if n >= 2:  # at least 2 of top-5 SAE features agree
                return top_cat  # e.g. "ligand-binding"

    return "unknown"

Step 6: Report

Cluster 1 — positions [12, 13]
  Structural: 0/2 interface, 2/2 ligand pocket (GTP), 0/2 core, all in P-loop helix
  UniProt:    Binding site (GTP) at residues 12, 13
              Domain: small GTPase
  SAE top 5:  ligand-binding (×2), secondary-structure (×3)
  → MECHANISM: ligand-binding (GTP P-loop)

Cluster 2 — positions [40, 41]
  Structural: 2/2 interface (chain B = RAF1-RBD)
  UniProt:    no specific annotation
  SAE top 5:  structural-stability (×3), domain (×2)
  → MECHANISM: interface (KRAS-RAF1 binding)

Step 7: Visualize — annotated DMS heatmap with hotspot callouts

The publication-style figure: DMS effect heatmap, sequence strip, structural annotation track, with per-hotspot mechanism callouts above the heatmap.

Align everything to one position axis. Heatmap column p, sequence letter p, every annotation bar covering residue p — all share x = p. Verify a landmark before drawing:

landmark_col = positions.index(12)  # column for KRAS pos 12
assert sequence[landmark_col] == "G", f"alignment broken at col {landmark_col}"

A 1-2 residue misalignment between heatmap and annotation track is a common, visually subtle error. If you've cross-joined two coordinate systems and any join was off-by-N, the whole figure is silently wrong. Verify here.

Heatmap + sequence + annotation track + callouts:

import matplotlib.pyplot as plt
import numpy as np

vlim = max(abs(np.nanmin(dms_matrix)), abs(np.nanmax(dms_matrix)))

fig, axes = plt.subplots(
    nrows=4, ncols=1, figsize=(max(8, 0.15 * len(positions)), 6),
    gridspec_kw={"height_ratios": [0.5, 4, 0.3, 0.5]}, sharex=True,
)
ax_callouts, ax_heat, ax_seq, ax_anno = axes

# Heatmap — symmetric diverging (RdBu_r); center on 0 for ΔΔG-style data
im = ax_heat.imshow(
    dms_matrix, aspect="auto", cmap="RdBu_r",
    vmin=-vlim, vmax=vlim,
    extent=(0, len(positions), 20, 0),
)
ax_heat.set_yticks(np.arange(20) + 0.5)
ax_heat.set_yticklabels(list(amino_acid_order))
ax_heat.set_ylabel("Substitution")

# Mark WT cells (box, no fill) — distinguish "WT" from "not measured"
for col, p in enumerate(positions):
    wt_aa = sequence[col]
    if wt_aa in amino_acid_order:
        row = amino_acid_order.index(wt_aa)
        ax_heat.add_patch(plt.Rectangle(
            (col, row), 1, 1, fill=False, edgecolor='black', linewidth=0.5,
        ))

# Sequence strip — one monospace letter per column
ax_seq.set_xlim(0, len(positions))
ax_seq.set_ylim(0, 1)
ax_seq.set_yticks([])
for col, letter in enumerate(sequence):
    ax_seq.text(col + 0.5, 0.5, letter, ha="center", va="center",
                family="monospace", fontsize=8)

# Annotation track — region colors (top half) + core bar (bottom half)
anno_by_pos = {a["position"]: a for a in struct["data"]["annotations"]}
region_colors = {"interface": "#1f77b4", "ligand": "#ff7f0e",
                 "both": "#2ca02c", "other": "#cccccc"}
for col, p in enumerate(positions):
    a = anno_by_pos.get(p, {})
    ax_anno.add_patch(plt.Rectangle(
        (col, 0.5), 1, 0.5, facecolor=region_colors.get(a.get("region", "other"), "#cccccc"),
    ))
    if a.get("is_core"):
        ax_anno.add_patch(plt.Rectangle((col, 0.0), 1, 0.5, facecolor="black"))
ax_anno.set_xlim(0, len(positions))
ax_anno.set_ylim(0, 1)
ax_anno.set_yticks([0.25, 0.75])
ax_anno.set_yticklabels(["core", "region"])
ax_anno.set_xlabel("Residue position")

# Callout row — per-hotspot mechanism boxes linked to clusters by brackets
for cluster, mechanism, top_features in hotspot_results:
    cluster_cols = [positions.index(p) for p in cluster if p in positions]
    if not cluster_cols:
        continue
    c_left, c_right = min(cluster_cols), max(cluster_cols)
    center = (c_left + c_right) / 2
    ax_heat.plot([c_left, c_right + 1], [0, 0], "k-", lw=2)
    label_lines = [f"MECHANISM: {mechanism}"] + [f"  {fl}" for fl in top_features[:3]]
    ax_callouts.text(center, 0.5, "\n".join(label_lines),
                     ha="center", va="center", fontsize=7,
                     bbox=dict(facecolor="white", edgecolor="black"))
    ax_callouts.plot([center, center], [0, -0.3], "k-", lw=0.5)
ax_callouts.set_xlim(0, len(positions))
ax_callouts.set_ylim(0, 1)
ax_callouts.axis("off")

fig.colorbar(im, ax=ax_heat, label="DMS effect (ΔΔG kcal/mol)")
plt.savefig("dms_hotspots_annotated.png", dpi=200, bbox_inches="tight")

Three cell-color rules to get right:

• Real measurement → diverging colour
• WT cell → boxed (the black outline above), value-cell colour = centre
• Not measured → distinct colour (e.g. light grey, not white — white reads

as "neutral" against the diverging palette)

Long proteins: for >300 residues, split into multiple horizontal panels (one panel per domain) rather than shrinking column width — the per-residue detail disappears below ~3 pixels per column.

Reproducing a published panel: verify its track alignment before treating it as ground truth. Published DMS panels do carry registration errors (the KRAS Fig 1i in the original paper is shifted +2 relative to its own sequence — see tooluniverse-protein-structural-annotation-pdb pitfalls).

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Interpretation table — what the mechanism call means downstream

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Honest limitations

1. Wrong PDB → wrong call. If your PDB doesn't include the relevant ligand or partner, the structural evidence layer is blind to that mechanism. Pick the structure that contains the right complex. 2. UniProt annotations are sparse for non-model proteins. Active sites are well-curated for canonical enzymes; novel proteins may have no annotated features and the skill falls back to structural + SAE only. 3. Single-position clusters limit statistical evidence. Descriptive ranking still works but permutation p-values can't (n=1). 4. SAE feature labels are interpretive hints, not ground truth. Labels come from how features activate across UniRef90, not per-protein expert curation. Treat "category: ligand-binding" as a hypothesis weight, not a proof. 5. Hotspots ≠ druggable sites. A catalytic residue is a critical residue but not necessarily a good drug target (allosteric pockets often are better). This skill explains why a residue is critical, not whether it's a good target. 6. The mechanism call is a synthesis of evidence, not a measurement. Don't quote the category as a fact — quote the evidence and the call as a reasoned conclusion.

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Cross-references