Detect distant homologs using profile and structure-aware methods that go beyond standard BLAST. Use when sequence identity falls into the twilight zone (<35% pairwise), when BLAST fails to find homologs that should exist, when working at metagenomic scale (DIAMOND, MMseqs2), or when structure beats sequence (Foldseek). Covers PSI-BLAST (iterative PSSM), jackhmmer (iterative HMM), HHblits/HHsearch (profile-profile), DIAMOND, MMseqs2, and Foldseek (3Di structural alphabet, van Kempen 2024).
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Detect distant homologs using profile and structure-aware methods that go beyond standard BLAST. Use when sequence identity falls into the twilight zone (<35% pairwise), when BLAST fails to find homologs that should exist, when working at metagenomic scale (DIAMOND, MMseqs2), or when structure beats sequence (Foldseek). Covers PSI-BLAST (iterative PSSM), jackhmmer (iterative HMM), HHblits/HHsearch (profile-profile), DIAMOND, MMseqs2, and Foldseek (3Di structural alphabet, van Kempen 2024).
Before using code patterns, verify installed versions match. If versions differ:
CLI: <tool> --version then <tool> --help to confirm flags
Python: pip show <package> then introspect signatures
If a flag is unrecognized or behavior changes, introspect with --help and adapt the example to match the installed version rather than retrying.
Remote Homology
"Find homologs my BLAST missed" -> Standard BLAST detects similarity reliably down to ~35% pairwise identity (the "twilight zone", Rost 1999 Protein Eng 12:85). Below that, profile methods (PSSMs, HMMs) and structure-aware methods (Foldseek) recover homologs that pairwise alignment misses.
This skill covers the decision: which method, when, against what database. The competition has shifted substantially since 2015: PSI-BLAST is no longer the de-facto standard; MMseqs2 and DIAMOND have replaced BLAST in most large-scale workflows; Foldseek (van Kempen et al. 2024 42:243) detects homologs no sequence method can reach by searching with a 3Di structural alphabet derived from AlphaFold/ESMFold predictions.
100-10,000x faster than BLAST at comparable sensitivity
Highest throughput, near-BLAST sensitivity
Identify distant protein homolog (single query)
jackhmmer
Iterative HMM; usually beats PSI-BLAST
Higher sensitivity than PSI-BLAST
Distant homology where structure available
Foldseek
3Di alphabet finds homologs sequence misses
Finds hits PSI-BLAST/HMMER cannot
Profile-profile comparison (PDB70 / Pfam)
HHblits + HHsearch
Profile vs profile is most sensitive when target also has profile
Best sensitivity for very-deep homology
Domain assignment
hmmscan against Pfam-A
Curated, calibrated thresholds
Standard practice
Metagenomic protein clustering
MMseqs2 easy-cluster
Scales to >1B sequences
Production-grade
ORF search vs metagenome
DIAMOND blastx --frameshift
Frameshift-aware; long reads
Best for noisy long reads
Structure-aware homology (no AF2 prediction available)
Foldseek + ProstT5
Predicts 3Di alphabet from sequence via PLM
Skip the AF2 step
Foldseek: the 2024 revolution
Foldseek (van Kempen, Kim, Tumescheit et al. 2024 Nat Biotechnol 42:243) searches protein structures by representing each residue's local geometry as a 21-letter "3Di" alphabet, then running BLAST-style alignment in this alphabet. Two consequences:
4-5 orders of magnitude faster than DALI (the previous gold-standard structure aligner).
Finds homologs that sequence methods cannot: when sequence has diverged past detection but structure is preserved, Foldseek recovers the homology. Reported sensitivity vs traditional structure search is comparable; sensitivity vs sequence methods is dramatically higher at low identity.
Two access modes:
Have a structure (PDB or AlphaFold): foldseek easy-search query.pdb db_dir result.m8 tmp_dir
Sequence only, no structure: use ProstT5 (Heinzinger et al. 2024) to embed sequence to 3Di alphabet directly, skipping AF2 entirely: foldseek databases ProstT5 prostt5_db tmp then foldseek easy-search seq.fa db result.m8 tmp --prostt5-model prostt5_db
The major prebuilt Foldseek databases (AlphaFoldDB, PDB100, ESMAtlas) are downloadable via foldseek databases.
PSI-BLAST: still useful, but watch the drift
PSI-BLAST (Altschul et al. 1997 Nucleic Acids Res 25:3389) builds a PSSM iteratively: each iteration includes hits below -inclusion_ethresh (default 0.005) in the next PSSM. Convergence is when no new hits cross the threshold. Stopping at convergence is often the wrong call -- iterations 2-3 are usually optimal; iterations 4+ frequently drift into paralog inclusion, contaminating the PSSM.
Parameter
Default
Postdoc tuning
-num_iterations
1
3 for most workflows; >3 risks drift
-inclusion_ethresh
0.005
0.002 if specificity matters (Altschul 1997 recommendation)
-evalue
10
0.01 for reporting cutoff
-num_threads
1
8 for large DBs
PSI-BLAST is also non-deterministic in detail: different input order or DB version can produce different PSSMs. For reproducibility, save the PSSM (-out_pssm pssm.asn) and re-use with -in_pssm.
HMMER 3 (hmmsearch, jackhmmer)
HMMER 3 (Eddy 2011 PLoS Comput Biol 7:e1002195) is profile HMM search. Two main workflows:
hmmsearch profile.hmm seqdb: search a database with a known HMM (Pfam, custom).
jackhmmer query.fa seqdb: iterative search like PSI-BLAST but with full HMM math. Typically higher sensitivity than PSI-BLAST at the same number of iterations.
For domain assignment, hmmscan query.fa Pfam-A.hmm is the canonical pipeline. Pfam HMMs come with calibrated gathering thresholds (-gathering) -- use them instead of arbitrary E-value cutoffs.
# Build domain database once
wget https://ftp.ebi.ac.uk/pub/databases/Pfam/current_release/Pfam-A.hmm.gz
gunzip Pfam-A.hmm.gz
hmmpress Pfam-A.hmm
# Annotate query against Pfam-A with gathering threshold (calibrated cutoff)
hmmscan --cut_ga --domtblout query.domtbl Pfam-A.hmm query.fa
HHblits / HHsearch (HH-suite3)
HHblits (Remmert et al. 2012 Nat Methods 9:173; HH-suite3: Steinegger et al. 2019 BMC Bioinformatics 20:473) is profile-profile alignment. The most sensitive method when both query and target have an HMM representation. Standard pipeline:
Build query MSA with hhblits against UniRef30 (or HHblits' default DB).
Convert MSA to query HMM.
Search against a profile DB (PDB70 for structure, Pfam-A for domains) with hhsearch.
Output is in HHM format. For very deep homology (the structural twilight zone), HHsearch vs PDB70 is still the gold standard.
MMseqs2 (the modern protein search workhorse)
MMseqs2 (Steinegger & Soding 2017 Nat Biotechnol 35:1026) replaces BLAST in nearly all large-scale workflows.
Key advantages:
Speed: 400-10,000x faster than blastp at similar sensitivity.
Sensitivity: At -s 7.5, matches HMMER sensitivity (but on raw sequence, not profiles).
Iterative profile search: mmseqs search --num-iterations 3 matches PSI-BLAST behavior at much higher speed.
The -s parameter trades sensitivity for speed: 1.0 (fast), 4.0 (default), 7.5 (HMMER-like sensitivity).
DIAMOND (the modern blastp replacement)
DIAMOND (Buchfink et al. 2015 Nat Methods 12:59; v2: Buchfink et al. 2021 Nat Methods 18:366) is the de-facto replacement for blastp on large-scale workflows.
For protein remote homology in 2026, DIAMOND --ultra-sensitive or MMseqs2 -s 7.5 should be the default before reaching for BLAST.
Iterative HMMER (jackhmmer)
# 3 iterations, save checkpoint HMM at each iteration
jackhmmer -N 3 --chkhmm iter.hmm --tblout hits.tbl query.fa uniref90.fa
# After convergence (no new hits below threshold) use the final HMM for downstream searches
hmmsearch iter-3.hmm target.fa > hits.txt
Code patterns
Foldseek search against AlphaFoldDB
Goal: Find structural homologs of a protein structure (or sequence via ProstT5) in AlphaFold's predicted structure database.
Approach: Download AlphaFoldDB structure DB (or ProstT5 for sequence-only); search with foldseek easy-search; parse m8 tabular output.
Reference (Foldseek 9+):
#!/bin/bash# Reference: foldseek 9+ | Verify API if version differsmkdir -p foldseek_dbs tmp
# Download the AlphaFoldDB Swiss-Prot subset (~few GB; full AFDB is much larger)
foldseek databases Alphafold/Swiss-Prot afdb_sp foldseek_dbs/tmp
# Structure-vs-structure search
foldseek easy-search query.pdb foldseek_dbs/afdb_sp results.m8 tmp \
--format-output query,target,fident,alnlen,evalue,bits,prob,qtmscore,ttmscore
head -5 results.m8
# qtmscore/ttmscore are TM-score equivalents from local Foldseek alignment.# Hits with prob > 0.9 are confidently structurally homologous.
This is the path to take when only a protein sequence is available -- ProstT5 (a protein language model) predicts the 3Di alphabet directly from sequence.
PSI-BLAST with saved PSSM
Goal: Build a position-specific scoring matrix iteratively, then re-use it for downstream searches.
Approach: 3 iterations against UniRef90 (or nr); save ASN.1 + ASCII PSSM; subsequent searches use -in_pssm.
Reference (NCBI BLAST+ 2.15+):
psiblast -query distant_protein.fa -db uniref90 \
-num_iterations 3 \
-inclusion_ethresh 0.002 \
-evalue 0.01 \
-num_threads 8 \
-out_pssm distant.pssm.asn \
-out_ascii_pssm distant.pssm.txt \
-out psiblast_results.txt
# Reuse saved PSSM in subsequent searches against a different DB
psiblast -in_pssm distant.pssm.asn -db swissprot \
-out swissprot_via_pssm.txt
MMseqs2 sensitive iterative search
Goal: PSI-BLAST-equivalent iterative profile search, but 100x faster.
Trigger: Iterating to convergence (5+ iterations).
Mechanism: Each iteration includes hits below threshold; eventually paralogs and divergent family members contaminate the PSSM.
Symptom: Later iterations return many implausible hits; functional inference goes wrong.
Fix: Cap at 3 iterations; inspect the saved PSSM and the included sequence set; use stricter -inclusion_ethresh 0.001.
Foldseek "structure but no homology" hits
Trigger: Searching small fragments or highly conserved folds (TIM barrels, Rossmann folds).
Mechanism: Structural fold is preserved across deep divergence; superfamily hits exist without true homology.
Symptom: High structural similarity to functionally unrelated proteins.
Fix: Combine Foldseek hits with sequence-based evidence; check shared catalytic residues; consider that fold-level similarity is necessary but not sufficient for homology.
MMseqs2 default sensitivity
Trigger:mmseqs easy-search without -s.
Mechanism: Default -s 4.0 is fast but misses remote homologs.
Symptom: Equivalent to a fast BLAST; misses what HMMER would find.
Fix: Set -s 7.5 for distant homology; -s 5.7 is a middle ground.
DIAMOND default mode lossy
Trigger:diamond blastp without --more-sensitive or --ultra-sensitive.
Mechanism: Default mode trades ~5% sensitivity for speed vs blastp.
Symptom: Hits BLAST would find are missing.
Fix: Use --more-sensitive for general work, --ultra-sensitive for remote homology.
Profile method on a low-complexity query
Trigger: Query has signal peptide, coiled-coil, or repeat region.
Mechanism: Profile is dominated by low-complexity columns; false hits to other low-complexity proteins.
Symptom: Many high-scoring hits to unrelated low-complexity proteins.
Fix: Mask the low-complexity region (SEG: segmasker -infmt fasta -in query.fa) before building the profile.
HHblits database version drift
Trigger: Using a UniRef30 database from a different release than the PDB70 search DB.
Mechanism: Profile statistics depend on the DB's amino acid distribution.
Symptom: Hit probabilities are miscalibrated.
Fix: Use UniRef30 and PDB70 from the same MMseqs2 / HH-suite release.
Foldseek without ProstT5 for sequence-only query
Trigger: No structure available; tried to predict with AF2 first (slow).
Mechanism: ProstT5 predicts 3Di alphabet directly from sequence, skipping AF2.
Symptom: Days-long AF2 prediction step for a query that could be Foldseek'd in seconds.
Fix: Use foldseek databases ProstT5 ... and --prostt5-model.
Common errors
Error / symptom
Cause
Solution
PSI-BLAST returns implausible hits
Profile drift (too many iterations)
Cap at 3 iterations; tighter -inclusion_ethresh
MMseqs2 hits all unrelated
Default sensitivity too low
-s 7.5
DIAMOND misses BLAST hits
Default mode lossy
--more-sensitive
Foldseek hits structurally unrelated proteins
Common fold, no homology
Cross-check with sequence and functional residues
HHblits prefilter no hits
Query MSA too sparse
Add -n 4 iterations; check input
jackhmmer ConvergenceError
Loop bug pre-v3.4
Upgrade HMMER
References
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389-3402.
Rost B. (1999) Twilight zone of protein sequence alignments. Protein Eng 12:85-94.
Remmert M, Biegert A, Hauser A, Soding J. (2012) HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods 9:173-175.
Buchfink B, Xie C, Huson DH. (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59-60.
Steinegger M, Soding J. (2017) MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat Biotechnol 35:1026-1028.
Steinegger M, Meier M, Mirdita M, Vohringer H, Haunsberger SJ, Soding J. (2019) HH-suite3 for fast remote homology detection and deep protein annotation. BMC Bioinformatics 20:473.
Buchfink B, Reuter K, Drost HG. (2021) Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods 18:366-368.
van Kempen M, Kim SS, Tumescheit C, Mirdita M, Lee J, Gilchrist CLM, Soding J, Steinegger M. (2024) Fast and accurate protein structure search with Foldseek. Nat Biotechnol 42:243-246.
Heinzinger M, Weissenow K, Sanchez JG, Henkel A, Mirdita M, Steinegger M, Rost B. (2024) Bilingual language model for protein sequence and structure. NAR Genom Bioinform 6:lqae150.
Related Skills
blast-searches - Remote BLAST against NCBI; baseline for closer homologs
local-blast - Local BLAST+ for moderate-scale workflows
