HH-suite

The HH-suite is an open-source software

package for sensitive protein sequence
HH-suite
searching. It contains programs that can search Developer(s) Johannes Söding, Michael
for similar protein sequences in protein sequence Remmert, Andreas Biegert,
databases. Sequence searches are a standard tool Andreas Hauser, Markus Meier,
in modern biology with which the function of Martin Steinegger
unknown proteins can be inferred from the
functions of proteins with similar sequences. Stable release 3.3.0 / 25 August 2020
HHsearch and HHblits are two main programs Repository github.com/soedinglab/hh-suite
in the package and the entry point to its search (https://github.com/soedinglab/hh
function, the latter being a faster iteration.[2][3] -suite)
HHpred is an online server for protein structure
prediction that uses homology information from Written in C++
HH-suite.[4] Operating system Unix-like; Debian package
available[1]
The HH-suite searches for sequences using
hidden Markov models (HMMs). The name Available in English
comes from the fact that it performs HMM- Type Bioinformatics tool
HMM alignments. Among the most popular License GPL v3
methods for protein sequence matching, the
programs have been cited more than 5000 times Website https://github.com/soedinglab/hh-
total according to Google Scholar.[5] suite

Background
Proteins are central players in all of life's processes. Understanding them is central to understanding
molecular processes in cells. This is particularly important in order to understand the origin of diseases. But
for a large fraction of the approximately 20 000 human proteins the structures and functions remain
unknown. Many proteins have been investigated in model organisms such as many bacteria, baker's yeast,
fruit flies, zebra fish or mice, for which experiments can be often done more easily than with human cells.
To predict the function, structure, or other properties of a protein for which only its sequence of amino acids
is known, the protein sequence is compared to the sequences of other proteins in public databases. If a
protein with sufficiently similar sequence is found, the two proteins are likely to be evolutionarily related
("homologous"). In that case, they are likely to share similar structures and functions. Therefore, if a protein
with a sufficiently similar sequence and with known functions and/or structure can be found by the
sequence search, the unknown protein's functions, structure, and domain composition can be predicted.
Such predictions greatly facilitate the determination of the function or structure by targeted validation
experiments.

Sequence searches are frequently performed by biologists to infer the function of an unknown protein from
its sequence. For this purpose, the protein's sequence is compared to the sequences of other proteins in
public databases and its function is deduced from those of the most similar sequences. Often, no sequences
with annotated functions can be found in such a search. In this case, more sensitive methods are required to
identify more remotely related proteins or protein families. From these relationships, hypotheses about the
protein's functions, structure, and domain composition can be inferred. HHsearch performs searches with a
protein sequence through databases. The HHpred server and the HH-suite software package offer many
popular, regularly updated databases, such as the Protein Data Bank, as well as the InterPro, Pfam, COG,
and SCOP databases.

Algorithm
Modern sensitive methods for protein search utilize sequence
profiles. They may be used to compare a sequence to a profile, or in
more advanced cases such as HH-suite, to match among
profiles.[2][6][7][8] Profiles and alignments are themselves derived
from matches, using for example PSI-BLAST or HHblits. A
position-specific scoring matrix (PSSM) profile contains for each
position in the query sequence the similarity score for the 20 amino
acids. The profiles are derived from multiple sequence alignments
(MSAs), in which related proteins are written together (aligned),
such that the frequencies of amino acids in each position can be
interpreted as probabilities for amino acids in new related proteins,
and be used to derive the "similarity scores". Because profiles
contain much more information than a single sequence (e.g. the
Iterative sequence search scheme
position-specific degree of conservation), profile-profile comparison of HHblits
methods are much more powerful than sequence-sequence
comparison methods like BLAST or profile-sequence comparison
methods like PSI-BLAST.[6]

HHpred and HHsearch represent query and database proteins by profile hidden Markov models (HMMs),
an extension of PSSM sequence profiles that also records position-specific amino acid insertion and
deletion frequencies. HHsearch searches a database of HMMs with a query HMM. Before starting the
search through the actual database of HMMs, HHsearch/HHpred builds a multiple sequence alignment of
sequences related to the query sequence/MSA using the HHblits program. From this alignment, a profile
HMM is calculated. The databases contain HMMs that are precalculated in the same fashion using PSI-
BLAST. The output of HHpred and HHsearch is a ranked list of database matches (including E-values and
probabilities for a true relationship) and the pairwise query-database sequence alignments.

HHblits, a part of the HH-suite since 2001, builds high-quality multiple sequence alignments (MSAs)
starting from a single query sequence or a MSA. As in PSI-BLAST, it works iteratively, repeatedly
constructing new query profiles by adding the results found in the previous round. It matches against a pre-
built HMM databases derived from protein sequence databases, each representing a "cluster" of related
proteins. In the case of HHblits, such matches are done on the level of HMM-HMM profiles, which grants
additional sensitivity. Its prefiltering reduces the tens of millions HMMs to match against to a few thousands
of them, thus speeding up the slow HMM-HMM comparison process.[3]

The HH-suite comes with a number of pre-built profile HMMs that can be searched using HHblits and
HHsearch, among them a clustered version of the UniProt database, of the Protein Data Bank of proteins
with known structures, of Pfam protein family alignments, of SCOP structural protein domains, and many
more.[9]

Applications
Applications of HHpred and HHsearch include protein structure prediction, complex structure prediction,
function prediction, domain prediction, domain boundary prediction, and evolutionary classification of
proteins.[10]
HHsearch is often used for homology modeling, that is, to build a model of the structure of a query protein
for which only the sequence is known: For that purpose, a database of proteins with known structures such
as the protein data bank is searched for "template" proteins similar to the query protein. If such a template
protein is found, the structure of the protein of interest can be predicted based on a pairwise sequence
alignment of the query with the template protein sequence. For example, a search through the PDB
database of proteins with solved 3D structure takes a few minutes. If a significant match with a protein of
known structure (a "template") is found in the PDB database, HHpred allows the user to build a homology
model using the MODELLER software, starting from the pairwise query-template alignment.

HHpred servers have been ranked among the best servers during CASP7, 8, and 9, for blind protein
structure prediction experiments. In CASP9, HHpredA, B, and C were ranked 1st, 2nd, and 3rd out of 81
participating automatic structure prediction servers in template-based modeling[11] and 6th, 7th, 8th on all
147 targets, while being much faster than the best 20 servers.[12] In CASP8, HHpred was ranked 7th on all
targets and 2nd on the subset of single domain proteins, while still being more than 50 times faster than the
top-ranked servers.[4]

Contents
In addition to HHsearch and HHblits, the HH-suite contains programs and perl scripts for format
conversion, filtering of MSAs, generation of profile HMMs, the addition of secondary structure predictions
to MSAs, the extraction of alignments from program output, and the generation of customized databases.

hhblits (Iteratively) search an HHblits database with a query sequence or MSA

hhsearch Search an HHsearch database of HMMs with a query MSA or HMM

hhmake Build an HMM from an input MSA

hhfilter Filter an MSA by maximum sequence identity, coverage, and other criteria

hhalign Calculate pairwise alignments, dot plots etc. for two HMMs/MSAs

reformat.pl Reformat one or many MSAs

addss.pl Add Psipred predicted secondary structure to an MSA or HHM file

hhmakemodel.pl Generate MSAs or coarse 3D models from HHsearch or HHblits results

hhblitsdb.pl Build HHblits database with prefiltering, packed MSA/HMM, and index files
multithread.pl Run a command for many files in parallel using multiple threads

splitfasta.pl Split a multiple-sequence FASTA file into multiple single-sequence files

renumberpdb.pl Generate PDB file with indices renumbered to match input sequence indices

The HMM-HMM alignment algorithm of HHblits and HHsearch was significantly accelerated using vector
instructions in version 3 of the HH-suite.[13]

See also
Sequence alignment software
Protein structure prediction
Position-specific scoring matrix
Multiple sequence alignment
CASP - Critical Assessment of Techniques for Protein Structure Prediction
 BLAST (Basic Local Alignment Search Tool)
Context-specific BLAST (CS-BLAST)

References
1. Debian hhsuite package (http://packages.debian.org/unstable/science/hhsuite)
2. Söding J (2005). "Protein homology detection by HMM-HMM comparison" (https://doi.org/10.
1093%2Fbioinformatics%2Fbti125). Bioinformatics. 21 (7): 951–960.
doi:10.1093/bioinformatics/bti125 (https://doi.org/10.1093%2Fbioinformatics%2Fbti125).
PMID 15531603 (https://pubmed.ncbi.nlm.nih.gov/15531603).
3. Remmert M, Biegert A, Hauser A, Söding J (2011). "HHblits: Lightning-fast iterative protein
sequence searching by HMM-HMM alignment" (http://wwwuser.gwdg.de/~compbiol/pdf/Prep
rint-2011-HHblits-inkl-Suppl2.pdf) (PDF). Nat. Methods. 9 (2): 173–175.
doi:10.1038/NMETH.1818 (https://doi.org/10.1038%2FNMETH.1818). hdl:11858/00-001M-
0000-0015-8D56-A (https://hdl.handle.net/11858%2F00-001M-0000-0015-8D56-A).
PMID 22198341 (https://pubmed.ncbi.nlm.nih.gov/22198341). S2CID 205420247 (https://ap
i.semanticscholar.org/CorpusID:205420247).
4. Söding J, Biegert A, Lupas AN (2005). "The HHpred interactive server for protein homology
detection and structure prediction" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC116016
9). Nucleic Acids Research. 33 (Web Server issue): W244–248. doi:10.1093/nar/gki408 (http
s://doi.org/10.1093%2Fnar%2Fgki408). PMC 1160169 (https://www.ncbi.nlm.nih.gov/pmc/art
icles/PMC1160169). PMID 15980461 (https://pubmed.ncbi.nlm.nih.gov/15980461).
5. Citations to HHpred (https://scholar.google.de/scholar?cluster=1978364893787489896), to
HHsearch (https://scholar.google.de/scholar?cluster=14407278965620614178), to HHblits
(https://scholar.google.de/scholar?cluster=12710838460360732088)
6. Jaroszewski L, Rychlewski L, Godzik A (2000). "Improving the quality of twilight-zone
alignments" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2144727). Protein Science. 9
(8): 1487–1496. doi:10.1110/ps.9.8.1487 (https://doi.org/10.1110%2Fps.9.8.1487).
PMC 2144727 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2144727). PMID 10975570
(https://pubmed.ncbi.nlm.nih.gov/10975570).
7. Sadreyev RI, Baker D, Grishin NV (2003). "Profile–profile comparisons by COMPASS
predict intricate homologies between protein families" (https://www.ncbi.nlm.nih.gov/pmc/arti
cles/PMC2366929). Protein Science. 12 (10): 2262–2272. doi:10.1110/ps.03197403 (https://
doi.org/10.1110%2Fps.03197403). PMC 2366929 (https://www.ncbi.nlm.nih.gov/pmc/article
s/PMC2366929). PMID 14500884 (https://pubmed.ncbi.nlm.nih.gov/14500884).
8. Dunbrack RL Jr (2006). "Sequence comparison and protein structure prediction". Current
Opinion in Structural Biology. 16 (3): 374–384. doi:10.1016/j.sbi.2006.05.006 (https://doi.org/
10.1016%2Fj.sbi.2006.05.006). PMID 16713709 (https://pubmed.ncbi.nlm.nih.gov/1671370
9).
9. Li, Zhaoyu. "Some Notes about HHSuite" (https://zhaoyu.li/post/hhsuite_notes/). Retrieved
3 April 2019.
10. Guerler A, Govindarajoo B, Zhang Y (2013). "Mapping Monomeric Threading to Protein–
Protein Structure Prediction" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076494).
Journal of Chemical Information and Modeling. 53 (3): 717–25. doi:10.1021/ci300579r (http
s://doi.org/10.1021%2Fci300579r). PMC 4076494 (https://www.ncbi.nlm.nih.gov/pmc/article
s/PMC4076494). PMID 23413988 (https://pubmed.ncbi.nlm.nih.gov/23413988).
11. Official CASP9 results for the template-based modeling category (121 targets) (http://predicti
oncenter.org/casp9/CD/data/html/groups.server.tbm.html)
12. Official CASP9 results for all 147 targets (http://predictioncenter.org/casp9/CD/data/html/grou
ps.2.html)
13. Steinegger M, Meier M, Mirdita M, Vöhringer H, Haunsberger S, Söding J (2019). "HH-suite3
for fast remote homology detection and deep protein annotation" (https://www.ncbi.nlm.nih.g
ov/pmc/articles/PMC6744700). BMC Bioinformatics. 20 (1): 473. doi:10.1186/s12859-019-
3019-7 (https://doi.org/10.1186%2Fs12859-019-3019-7). PMC 6744700 (https://www.ncbi.nl
m.nih.gov/pmc/articles/PMC6744700). PMID 31521110 (https://pubmed.ncbi.nlm.nih.gov/31
521110).

External links
Soeding Lab (http://www.mpibpc.mpg.de/soeding) at Max-Planck Institute in Göttingen - HH-
suite developers
Precompiled HH-suite binaries and databases (http://wwwuser.gwdg.de/~compbiol/data/hhs
uite/) download from developers
HHpred (http://arquivo.pt/wayback/20160514083149/http%3A//toolkit.tuebingen.mpg.de/hhp
red) — free server at Max-Planck Institute in Tuebingen
HHblits (http://toolkit.tuebingen.mpg.de/hhblits) — free server at Max-Planck Institute in
Tuebingen
CASP website (http://predictioncenter.org/)
CASP9 template-based modeling results (http://predictioncenter.org/casp9/groups_analysis.
cgi?type=server&tbm=on&tbmfm=on&submit=Filter)
HH-suite debian package (https://packages.debian.org/de/sid/science/hhsuite)
HH-suite ubuntu package (http://packages.ubuntu.com/raring/science/hhsuite)
HH-suite arch linux user repository (https://aur.archlinux.org/packages/hhsuite)

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