Troubleshooting & Common Pitfalls
A quick-lookup page for "something seems off" moments — organized by symptom, not by tool. Each row links to the page with the full explanation; this page is for finding the right row fast, not for reading start to finish.
Reading a prediction
| Symptom |
Likely cause |
Where to read more |
| A region looks confidently folded (high pLDDT) but you know it's disordered |
Conditionally-folded IDR — AlphaFold can predict the bound or modified conformation with high confidence even from a naked sequence |
pLDDT is not a clean disorder detector |
| A clean-looking helix has low pLDDT |
Probably a real signal, not noise — likely a transient/conditional helix, or (in AlphaFold3 specifically) a hallucinated one |
Confidence Metrics |
| Two domains each look great individually, but you're not sure about their arrangement |
pLDDT can't tell you — that's what PAE is for |
Why do I need PAE if I already have pLDDT? |
| Two chains are touching in the 3D view — are they really interacting? |
Not necessarily. AlphaFold/ColabFold always places chains near each other in a multimer job, even with no real interaction |
Chance or determination? |
| ipTM varies a lot across the 5 models for the same complex |
Not a bug — can be a real signal of interface ambiguity, or one outlier among four agreeing models |
Real-world example |
| You mutated a residue in silico and pLDDT barely changed (or moved the "wrong" way) |
Expected — AF2 is largely insensitive to point mutations; pLDDT often doesn't track real stability effects |
What Actually Influences a Prediction? |
| A PTM you specified doesn't seem to change anything |
You're using AlphaFold2/ColabFold — they have no PTM representation at all. Only AlphaFold3 supports PTMs as explicit input |
What Actually Influences a Prediction? |
AlphaMissense
| Symptom |
Likely cause |
Where to read more |
| AlphaMissense fetch returns nothing for your protein |
It's not human, or not the canonical sequence — scores only exist natively for human canonical UniProt sequences |
Using AlphaMissense on a non-human protein |
| Residue numbering seems off vs. what you expected from AlphaFold DB |
You may be looking at an isoform-specific entry, or a canonical one when you needed an isoform (or vice versa) |
Check which sequence you're actually looking at |
| A score seems too extreme / doesn't match known clinical classification |
AlphaMissense over-predicts pathogenicity in some benchmarks and is supporting evidence only, never standalone |
Known limitations |
ChopChopMF
| Symptom |
Likely cause |
Fix |
| Crop/Delete removed too much and can't be undone |
No duplicate made first |
Always Duplicate Structure before a destructive edit — see Trimming a model |
| PAE Contacts tool won't run / gives odd results |
More than one model open |
Close/hide other models — only one is supported |
| Foldseek hits look noisy or miss the obvious homolog |
Flexible/disordered regions still in the model, or cropped down to a single helix/sheet |
Crop a complete domain first — see Finding structural homologs |
| Foldseek finds nothing for a poorly characterized organism |
Searched PDB only |
Switch the target database to AlphaFold DB (afdb50) |
| ΔG plot dominated by tiny values |
Neutral band toggle off |
Leave Neutral band (±ε) checked |
Running ColabFold / HPC
| Symptom |
Likely cause |
Where to read more |
| A run is spending most of its time before inference even starts |
Expected — MSA search (not the network) is the classic AF2 bottleneck, historically ~80%+ of wall-clock time |
AF2 vs. ColabFold: same brain, faster search |
| AlphaFold3 container won't run on an older cluster |
CUDA version mismatch — AF3 needs CUDA 12.3+, older cluster drivers may only support 12.2 |
Running ColabFold (local/HPC) — containerize with Apptainer |
| AlphaFold Server rejects a job or a ligand/ion |
Server enforces a 5,000-token cap and only supports a specific ion list (MG, ZN, CL, CA, NA, MN, K, FE, CU, CO) |
AlphaFold3 & AlphaFold Server practical limits |
Still stuck?
Check Resources & Further Reading for primary sources, or — for the tools themselves rather than this guide's explanations — report it directly where they're maintained: ChopChopMF issues · ChimeraX-FigureStyle issues.