Protein design qc

Protein Design Quality Control And Ranking

Plain-language role: Use this skill to decide which designs pass QC and which ones should move forward.

Critical Limitation

Individual metrics have weak predictive power for binding. Research shows:

• Individual metric ROC AUC: 0.64-0.66 (slightly better than random)
• Metrics are pre-screening filters, not affinity predictors
• Composite scoring is essential for meaningful ranking

These thresholds filter out poor designs but do NOT predict binding affinity.

QC Organization

QC is organized by purpose and level:

Purpose	What it assesses	Key metrics
Binding	Interface quality, binding geometry	ipTM, PAE, SC, dG, dSASA
Expression	Manufacturability, solubility	Instability, GRAVY, pI, cysteines
Structural	Fold confidence, consistency	pLDDT, pTM, scRMSD

Each category has two levels:

• Metric-level: Calculated values with thresholds (pLDDT > 0.85)
• Design-level: Pattern/motif detection (odd cysteines, NG sites)

---

Quick Reference: All Thresholds

Category	Metric	Standard	Stringent	Source
Structural	pLDDT	> 0.85	> 0.90	AF2/Chai/Boltz
	pTM	> 0.70	> 0.80	AF2/Chai/Boltz
	scRMSD	< 2.0 Å	< 1.5 Å	Design vs pred
Binding	ipTM	> 0.50	> 0.60	AF2/Chai/Boltz
	PAE_interaction	< 12 Å	< 10 Å	AF2/Chai/Boltz
	Shape Comp (SC)	> 0.50	> 0.60	PyRosetta
	interface_dG	< -10	< -15	PyRosetta
Expression	Instability	< 40	< 30	BioPython
	GRAVY	< 0.4	< 0.2	BioPython
	ESM2 PLL	> 0.0	> 0.2	ESM2

Design-Level Checks (Expression)

Pattern	Risk	Action
Odd cysteine count	Unpaired disulfides	Redesign
NG/NS/NT motifs	Deamidation	Flag/avoid
K/R >= 3 consecutive	Proteolysis	Flag
>= 6 hydrophobic run	Aggregation	Redesign

See: references/binding-qc.md, references/expression-qc.md, references/structural-qc.md

---

Sequential Filtering Pipeline

import pandas as pd

designs = pd.read_csv('designs.csv')

# Stage 1: Structural confidence
designs = designs[designs['pLDDT'] > 0.85]

# Stage 2: Self-consistency
designs = designs[designs['scRMSD'] < 2.0]

# Stage 3: Binding quality
designs = designs[(designs['ipTM'] > 0.5) & (designs['PAE_interaction'] < 10)]

# Stage 4: Sequence plausibility
designs = designs[designs['esm2_pll_normalized'] > 0.0]

# Stage 5: Expression checks (design-level)
designs = designs[designs['cysteine_count'] % 2 == 0]  # Even cysteines
designs = designs[designs['instability_index'] < 40]

---

Composite Scoring (Required for Ranking)

Individual metrics alone are too weak. Use composite scoring:

def composite_score(row):
    return (
        0.30 * row['pLDDT'] +
        0.20 * row['ipTM'] +
        0.20 * (1 - row['PAE_interaction'] / 20) +
        0.15 * row['shape_complementarity'] +
        0.15 * row['esm2_pll_normalized']
    )

designs['score'] = designs.apply(composite_score, axis=1)
top_designs = designs.nlargest(100, 'score')

For advanced composite scoring, see references/composite-scoring.md.

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Tool-Specific Filtering

BindCraft Filter Levels

Level	Use Case	Stringency
Default	Standard design	Most stringent
Relaxed	Need more designs	Higher failure rate
Peptide	Designs < 30 AA	~5-10x lower success

BoltzGen Filtering

boltzgen run ... \
  --budget 60 \
  --alpha 0.01 \
  --filter_biased true \
  --refolding_rmsd_threshold 2.0 \
  --additional_filters 'ALA_fraction<0.3'

• alpha=0.0: Quality-only ranking
• alpha=0.01: Default (slight diversity)
• alpha=1.0: Diversity-only

---

Design-Level Severity Scoring

For pattern-based checks, use severity scoring:

Severity Level	Score	Action
LOW	0-15	Proceed
MODERATE	16-35	Review flagged issues
HIGH	36-60	Redesign recommended
CRITICAL	61+	Redesign required

---

Experimental Correlation

Metric	AUC	Use
ipTM	~0.64	Pre-screening
PAE	~0.65	Pre-screening
ESM2 PLL	~0.72	Best single metric
Composite	~0.75+	Always use

Key insight: Metrics work as filters (eliminating failures) not predictors (ranking successes).

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Campaign Health Assessment

Quick assessment of your design campaign:

Pass Rate	Status	Interpretation
> 15%	Excellent	Above average, proceed
10-15%	Good	Normal, proceed
5-10%	Marginal	Below average, review issues
< 5%	Poor	Significant problems, diagnose

---

Failure Recovery Trees

Too Few Pass pLDDT Filter (< 5% with pLDDT > 0.85)

Low pLDDT across campaign
├── Check scRMSD distribution
│   ├── High scRMSD (>2.5Å): Backbone issue
│   │   └── Fix: Regenerate backbones with lower noise_scale (0.5-0.8)
│   └── Low scRMSD but low pLDDT: Disordered regions
│       └── Fix: Check design length, simplify topology
├── Try more sequences per backbone
│   └── modal run modal_proteinmpnn.py --num-seq-per-target 32 --sampling-temp 0.1
├── Use SolubleMPNN instead of ProteinMPNN
│   └── Better for expression-optimized sequences
└── Consider different design tool
    └── BindCraft (integrated design) may work better

Too Few Pass ipTM Filter (< 5% with ipTM > 0.5)

Low ipTM across campaign
├── Review hotspot selection
│   ├── Are hotspots surface-exposed? (SASA > 20Ų)
│   ├── Are hotspots conserved? (check MSA)
│   └── Try 3-6 different hotspot combinations
├── Increase binder length (more contact area)
│   └── Try 80-100 AA instead of 60-80 AA
├── Check interface geometry
│   ├── Is target flat? → Try helical binders
│   └── Is target concave? → Try smaller binders
└── Try all-atom design tool
    └── BoltzGen (all-atom, better packing)

High scRMSD (> 50% with scRMSD > 2.0Å)

Sequences don't specify intended structure
├── ProteinMPNN issue
│   ├── Lower temperature: --sampling-temp 0.1
│   ├── Increase sequences: --num-seq-per-target 32
│   └── Check fixed_positions aren't over-constraining
├── Backbone geometry issue
│   ├── Backbones may be unusual/strained
│   ├── Regenerate with lower noise_scale (0.5-0.8)
│   └── Reduce diffuser.T to 30-40
└── Try different sequence design
    └── ColabDesign (AF2 gradient-based) may work better

Everything Passes But No Experimental Hits

In silico metrics don't predict affinity
├── Generate MORE designs (10x current)
│   └── Computational metrics have high false positive rate
├── Increase diversity
│   ├── Higher ProteinMPNN temperature (0.2-0.3)
│   ├── Different backbone topologies
│   └── Different hotspot combinations
├── Try different design approach
│   ├── BindCraft (different algorithm)
│   ├── ColabDesign (AF2 hallucination)
│   └── BoltzGen (all-atom diffusion)
└── Check if target is druggable
    └── Some targets are inherently difficult

Too Many Designs Pass (> 50%)

Suspiciously high pass rate
├── Check if thresholds are too lenient
│   └── Use stringent thresholds: pLDDT > 0.90, ipTM > 0.60
├── Verify prediction quality
│   ├── Are predictions actually running? Check output files
│   └── Are complexes being predicted, not just monomers?
├── Check for data issues
│   ├── Same sequence being predicted multiple times?
│   └── Wrong FASTA format (missing chain separator)?
└── Apply diversity filter
    └── Cluster at 70% identity, take top per cluster

---

Diagnostic Commands

Quick Campaign Assessment

import pandas as pd

df = pd.read_csv('designs.csv')

# Pass rates at each stage
print(f"Total designs: {len(df)}")
print(f"pLDDT > 0.85: {(df['pLDDT'] > 0.85).mean():.1%}")
print(f"ipTM > 0.50: {(df['ipTM'] > 0.50).mean():.1%}")
print(f"scRMSD < 2.0: {(df['scRMSD'] < 2.0).mean():.1%}")
print(f"All filters: {((df['pLDDT'] > 0.85) & (df['ipTM'] > 0.5) & (df['scRMSD'] < 2.0)).mean():.1%}")

# Identify top issue
if (df['pLDDT'] > 0.85).mean() < 0.1:
    print("ISSUE: Low pLDDT - check backbone or sequence quality")
elif (df['ipTM'] > 0.50).mean() < 0.1:
    print("ISSUE: Low ipTM - check hotspots or interface geometry")
elif (df['scRMSD'] < 2.0).mean() < 0.5:
    print("ISSUE: High scRMSD - sequences don't specify backbone")

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Templates and Demo

• Executable scorer: scripts/protein_qc_score.py
• Sample metrics table: templates/protein-design-qc/design_metrics.csv
• Demo input table: examples/minimal-binder-campaign/inputs/design_metrics.csv

Inputs

• A CSV table of design metrics such as pLDDT, ipTM, PAE, scRMSD, and optional ESM or expression fields.
• Optional threshold overrides for stricter or more permissive filtering.
• A design campaign context so summary pass rates can be interpreted correctly.

Outputs

• Per-design pass or fail annotations across structural, binding, and expression checks.
• A composite score and ranked output table suitable for experimental triage.
• A campaign summary highlighting which filter stage is removing most candidates.

Next Step

Advance the top-ranked designs into ipsae or experimental testing, and use the summary to decide whether the generation stage needs adjustment.