complex-formation

name	complex-formation
description	Assemble and purify macromolecular complexes for structural biology. Use when the user asks about gel filtration, complex assembly, SEC purification, elongation complexes, calculating stoichiometry, or interpreting A260/A280 ratios.
allowed-tools	Bash(complex_cli:*)

Complex Formation Skill

Guidelines for assembling and purifying macromolecular complexes for structural biology.

Gel Filtration (Size Exclusion Chromatography)

Standard Setup

Parameter	Value
Column	Superose 6 Increase 3.2/300
System	ÄKTA micro
Fraction size	0.05 mL (50 µL)
Void volume	B6

Elution Positions by Complex Size

Complex	Expected Fractions	Notes
Large EC* (full elongation complex)	B4-B5	DSIF, SPT6, PAF1c, etc.
EC* + nucleosome	B5-B6	May approach void
Minimal EC (Pol II + DSIF)	B5-C3	Intermediate
Pol II only	C4-C6	~500 kDa reference

Rule: If complex elutes earlier than expected → possible aggregation. If later → incomplete assembly or dissociation.

A260/A280 Ratio Guidelines

Complex Type	Expected Ratio	Interpretation
EC* on linear DNA	A280 > A260	Protein dominates signal
Nucleosome-containing	A260 >> A280	Nucleosomal DNA dominates

Red flags:

• A260/A280 inverted from expectation → wrong complex or contamination
• Very high A260 → free nucleic acid contamination

Concentration Targets

Application	Target Range	Minimum
Cryo-EM grids	100-200 nM	60 nM
Transcription elongation assays	100-200 nM	60 nM

Below 60 nM: Not usable — concentration too low for downstream applications.

Quality Control Checkpoints

1. GF Trace Analysis

• Check elution volume matches expected MW
• Verify A260/A280 ratio is appropriate for complex type
• Look for symmetric peak (asymmetry suggests heterogeneity)

2. SDS-PAGE

• Always run SDS-PAGE on fractions before proceeding
• Check for:
  • All expected subunits present
  • Correct stoichiometry (band intensities)
  • No unexpected bands (contamination/degradation)
  • No missing subunits (incomplete assembly)

3. Fraction Selection

• Use single fractions (do not pool)
• Selected fractions get cross-linked before grid preparation

Complex Assembly Workflow

1. Prepare components — Query LabBook for current stock concentrations
2. Calculate recipe — Use complex_cli.py with appropriate preset
3. Mix components — Follow stoichiometry from preset (typically 1.5× excess over Pol II)
4. Incubate — Allow complex formation (timing depends on complex)
5. GF purification — Superose 6 Increase 3.2/300
6. Analyze fractions — Check trace + run SDS-PAGE
7. Select fraction — Based on elution position, A260/A280, gel appearance
8. Cross-link — If proceeding to cryo-EM
9. Grid preparation — At 100-200 nM

CLI Tools

# List available presets
python3 scripts/complex_cli.py list

# Calculate recipe for EC*
python3 scripts/complex_cli.py ec_star --pol2 5.2uM --volume 100

# Calculate TC-NER complex
python3 scripts/complex_cli.py tc_ner --pol2 5.2uM --volume 100

Troubleshooting

Problem	Possible Cause	Solution
Elutes at void (B6)	Aggregation	Reduce concentration, add salt, check buffer
Elutes later than expected	Incomplete assembly	Check component activity, increase incubation
Missing bands on gel	Component limiting	Verify stock concentrations, check stoichiometry
Extra bands on gel	Contamination/degradation	Check protein quality, use fresh stocks
Low A280 signal	Low yield	Scale up input, optimize assembly conditions

Empirical Knowledge

Column calibration is empirical — based on previous runs with known complexes. No formal MW standards are used; positions are learned from experience.

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Last updated: 2026-01-17