Online Sources of Sequence Alignments
This is by no means an exhaustive list, but includes several sources of
protein multiple sequence alignments for use in
Why you might want such alignments:
Chimera(X) can be used to display the sequence alignment,
calculate and display sequence conservation,
superimpose structures, morph between structures, etc.
- you are interested in a particular set of proteins
and want to map sequence conservation onto structure(s)
- you have a protein structure and want to find other structures
in the same family or superfamily and compare them
Related tutorials (Chimera):
Sequences and Structures,
Superpositions and Alignments,
Mapping Sequence Conservation onto Structures
Related tutorials (ChimeraX):
Coloring by Sequence Conservation
*Chimera(X) is not meant to handle very large multiple sequence alignments
(those containing several hundreds to thousands of sequences).
A good tool for working with large alignments is
It allows redundancy-filtering and saving a smaller alignment in
various formats (aligned FASTA, etc.) that can be read into Chimera.
Jalview also calculates per-column conservation directly and via
web service; these annotation values can be exported as CSV,
but it can be difficult to reformat them properly into a
sequence alignment header file
(with the same number of positions as the filtered alignment) or
residue attribute file for use in Chimera.
A direct connection between Jalview and Chimera is under development.
Alignment sources are grouped as follows:
Descriptions here are minimal; see literature references and/or
other documentation at the individual websites for more details.
Points to consider:
- How diverse a set of sequences is appropriate for your purposes?
Must the proteins have identical functions, or are similar structures and
related functions sufficient?
Be aware that different sources have different definitions of
family and superfamily.
- How many related structures are known?
It may be more convenient for sequence-structure work to use
sequence alignments of known structures
but that is only possible when several structures have been solved.
If only a single structure (perhaps even a homology model) is available,
you will have to use at least some sequences without known structures
to calculate conservation.
- One advantage of using precalculated alignments from a database,
or an alignment calculated by a server such as
is that in your own published work, you can cite the source rather than
having to describe in detail and justify how you generated your own alignment.
These databases contain sequence alignments of proteins with experimentally
determined 3D structures. Typically the names in the alignment are
structure identifiers, which makes it easy to autofetch all the structures with
a single step in Chimera (from the sequence alignment window, choose
Structure... Load Structures). Of course, you can just fetch
a subset of the structures individually with the open command or
File... Fetch by ID.
- alignments are for families, generally narrow sets of proteins
with high similarities
- download *.pir or *.ali sequence alignment (either variant of
aligned NBRF/PIR format is accepted by Chimera(X))
- can also download a coordinate file in which each structure is a
different chain; this is not as convenient as separate models in Chimera(X),
so I prefer to fetch structures as described above, then superimpose
them using the sequence alignment
- can autofetch structures from the PDB since the sequences
are named with PDB IDs
Tip: getting rid of extra chains
Often structures include additional chains that are not associated with
the sequence alignment and not needed for the intended analyses. These
chains may be additional copies of the same protein or different
macromolecules. Here is a trick for removing such unassociated chains
- With the mouse in the sequence alignment, draw a box that includes
at least one associated residue from each structure.
That will select the associated residues.
- Click into the main graphics window and press the keyboard up arrow
key to promote the selection from residues to chains.
- Invert the selection to contain unwanted atoms:
Command: sel invert
Menu: Select... Invert (all models)
- Delete the selection:
Command: del sel
Menu: Actions... Atoms/Bonds... delete
- structure-based alignments of
superfamilies filtered at 40% sequence identity (SCOPe release 2.06)
- *.ali files (aligned NBRF/PIR format)
- superimposed structures can also be downloaded
- PASS2 sequence names can be converted to SCOP IDs
(e.g. for Chimera fetch by ID) by changing any “-”
(hyphen) to “_” (underscore)
If the corresponding tree in New Hampshire (aka Newick) format is available,
it can be loaded after the sequence alignment has been opened.
- PFAM (also tree files)
- for Chimera purposes, I use “seed alignments”
(many fewer sequences than the full PFAM alignments)
- don't get their MSF format (last time I checked, it was wrong),
but Selex, Stockholm, FASTA, and the tree format are all fine
- for some families, corresponding structures are listed;
sometimes structures are not listed even though they exist
- can link out to PFAM from structure pages at the RCSB PDB
manual sequence-structure association
- Often your structure will not be similar enough to any sequence in the
alignment to associate automatically. Some things to try to associate
- Use Structure... Associations in Multalign Viewer to
compare all the sequences to the structure and associate it with the best
match even though it does not meet the automatic association criteria.
The resulting association must be examined, since it may not be good
enough to be usable. For example, the forced association of structure
2gbp with PF00532_seed.slx
only associates the first few residues of the structure with the
last few residues of a sequence and is obviously wrong.
- Use Edit... Add Sequence in Multalign Viewer and add
the sequence from the structure to the alignment. This may require several
cycles of unsuccessfully adding the sequence, using Edit... Delete
Sequences/Gaps to remove it, and adding it back again with different
alignment parameters. For example, adding the sequence of
2gbp to PF00532_seed.slx does not work with the default
parameters, but is reasonably successful using the BLOSUM-30 matrix.
(Simple Modular Architecture Research Tool)
alignments for >1000 domain families
- domain pages allow viewing alignments in various formats
- for Chimera purposes, I showed MSF format and then used cut-and-paste
to save the browser contents to a plain text file named *.msf
(had problems with their FASTA; didn't try other formats)
- domain page Structure section lists corresponding PDB entries
- NCBI CDD
(Conserved Domain Database)
domain-based local multiple sequence alignments from multiple
sources including PFAM, SMART, COGs, TIGRFAM, PRK, and NCBI-curated
- choose number of sequences (Row Display), choices range from 10-100
- specify how they are chosen (Type Selection),
default “the most diverse members”
- choose format mFasta, click Reformat, then copy-and-paste
to create a plain text file named *.fasta
(or one of the other suffixes recognized by Chimera as aligned FASTA)
(Structure-Function Linkage Database)
family, subgroup, and superfamily sequence alignments,
Chimera “active site” sessions for some families
- detailed information but for relatively few enzyme superfamilies
- lists some but not all structures
- can set up browser (except Safari) to open structures, sequence
alignments, sessions directly in Chimera
- can get an alignment that includes your query sequence if it matches HMMs
in the database
- ConSurf (also creates tree files)
- the input is one structure
- you can get precalculated results for PDB entries or submit a structure
to the server (server calculations may take a while)
- the server has many options; if you just use a single structure as input,
it will find homologous sequences and create a sequence alignment and tree
- alternatively, you can upload an existing multiple sequence alignment
(several formats accepted) for conservation calculations
- results available for download include the sequence alignment
(Clustal *.aln format), per-column ConSurf conservation scores,
and TheTree.txt (tree in Newick format)
- can set up browser (except Safari) to
show results directly in Chimera
- the Blast Protein tool in Chimera
(under Tools... Sequence in the main menu,
under Info in the Multalign Viewer menu)
uses a web service hosted by the
As with any BLAST search,
the results depend on the search criteria and the database, and
may be unbalanced and/or contain significant redundancy.
Be aware that BLAST is meant to find similar sequences quickly rather than to
generate accurate alignments (more on this below).
- the input is one sequence, any of the following:
- a chain from a structure open in Chimera
- a sequence pasted in as text
- a sequence from an alignment open in Chimera's
- several parameters can be adjusted, including number of iterations
(multiple iterations = PSI-BLAST) and database (pdb or nr)
- the output is a list of hits, from which all or a user-chosen subset
can be retrieved as:
- a pseudo-multiple sequence alignment,
automatically shown in Chimera's
A pseudo-multiple alignment from BLAST is not a true multiple alignment,
but a consolidation of the pairwise alignments of individual hits to the
query, as specified by the BLAST
alignment view option
“flat query-anchored with letters for identities.”
Further, Blast may omit parts of the hit sequences. See tips below.
- structures for hits from pdb, automatically superimposed
according to the pseudo-multiple sequence alignment
Tips: improving a Blast pseudo-multiple alignment
- It may be helpful to realign the sequences with true multiple-alignment
software (e.g., using
Edit... Realign Sequences
in the Multalign Viewer menu)
- To fix the problem of omitted residues,
if structures for the hit sequences have been loaded in Chimera,
a new multiple alignment of their full sequences can be generated with
Align Chain Sequences
(under Tools... Sequence in the main Chimera menu).
Issues to consider are how diverse the set of sequences should be,
alignment quality, and balance, i.e. an alignment could oversample
some areas of the intended “sequence space” and undersample others.
Imbalance can be reduced by
filtering out sequences at some level of sequence identity, and in Chimera,
using sequence-weighting options to calculate conservation.
I used the DIY approach to make the alignments in the Chimera
demo (under Tools... Demos in the menu)
because I wanted to include sequences for the hormone and receptor
from the same six species.
The sequences were similar enough to align easily, so I didn't have
to worry about tweaking parameters to improve the results.
Look up sequences (I usually save or text-edit the sequences into
a single FASTA file):
Use a server to align them (order is merely alphabetical):
December 2020 / meng[at]cgl.ucsf.edu /
If you find broken links or outdated information in this page,
please let me know – thanks!