A protein family from a list of accessions

Human NLRP1, an inflammasome sensor, starts with a pyrin domain (PYD) of about eighty residues that the mouse protein lacks. Aligned with its orthologs, the domains every vertebrate copy shares fall in one band of columns each, and a missing PYD shows as blank space. For twelve NLRP1 orthologs named by UniProt accession, we:

fetch the sequences and align them
infer a tree from the alignment
look up the Pfam domains in InterPro and open the three files together
filter the overlay to the PYD and check it against the GFF
share the view as one link

Prerequisites

curl
ClustalW, apt install clustalw on Debian or Ubuntu, brew install clustal-w on macOS. ClustalW both aligns and infers a tree, so this page needs no other aligner.
react-msaview-cli, npm install -g react-msaview-cli, NodeJS v22+

The data

Twelve NLRP1 orthologs as UniProtKB holds them, with Pfam matches from InterPro release 110.0. Nothing to download: the commands below fetch these.

What it is	File
One protein sequence per accession, for example	Q9C000.fasta
Precomputed Pfam matches per accession, for example	Q9C000

The files this page writes, hosted for the figure links

What it is	File
The twelve sequences before alignment, right-padded to one width	nlrp1-unaligned.aln
The alignment	nlrp1.aln
Its tree	nlrp1.nh
Its domains	nlrp1-domains.gff

1. Name the rows

Write one accession per line, followed by the label you want down the side of the viewer. The label becomes the FASTA defline, the tree tip, and the first column of the domain GFF.

Q9C000	Human
H2QC06	Chimp
A0A1D5QWR0	Rhesus
A0A8I3MJ75	Dog
A0ABM3YGI7	Hedgehog
E1BNN6	Cow
K9IW94	Pig
A0A9L0RFW2	Horse
Q2LKU9	Mouse
D9I2G4	Rat
A0ABM2XMM7	Hamster
A0A386CAB9	Zebrafish

Save that as accessions.tsv, with a tab between the two columns.

2. Fetch the sequences

# read each accession and write one FASTA record named by its label, which is
# what the viewer draws down the side
while IFS=$'\t' read -r accession label; do
  printf '>%s\n' "$label" >> family.fasta
  curl -sf "https://rest.uniprot.org/uniprotkb/$accession.fasta" |
    tail -n +2 | tr -d '\n' >> family.fasta
  printf '\n' >> family.fasta
done < accessions.tsv

tail -n +2 drops UniProt’s defline, and tr -d '\n' unwraps the sequence onto one line. Check the lengths before aligning:

awk '/^>/{name=$0; next}{print name, length($0)}' family.fasta

>Human 1473
>Chimp 1471
>Rhesus 1537
>Dog 1432
>Hedgehog 1360
>Cow 1410
>Pig 1294
>Horse 1465
>Mouse 1182
>Rat 1218
>Hamster 1143
>Zebrafish 1355

A truncated fragment or a stray isoform would show here as an outlier length. The viewer opens unaligned sequences once every row is the same length, so right-padding the shorter ones loads family.fasta as twelve rows.

The twelve sequences before alignment, colored by residue, with a ragged right edge where each row ends. The conservation track above is near flat.

3. Align them

clustalw -INFILE=family.fasta -ALIGN -TYPE=PROTEIN \
  -OUTPUT=FASTA -OUTFILE=family.afa

ClustalW finishes in under two seconds and reports Alignment Score 258590 over 1666 columns, 129 more than the longest input.

The twelve sequences after ClustalW, at 1666 columns. Vertical bands of color run through every row where the rows share residues, the pale stretches are gaps ClustalW inserted, and the conservation track now rises and falls.

4. Infer a tree

# -TREE reads an existing alignment and writes neighbor-joining Newick
clustalw -INFILE=family.afa -TREE -TYPE=PROTEIN -OUTPUTTREE=phylip

# ClustalW wraps the Newick across many lines; the viewer wants one string
tr -d '[:space:]' < family.ph > family.nwk

The Newick puts Zebrafish inside the rodents:

(((((Mouse:0.11672,Rat:0.11797):0.05644,Hamster:0.14779):0.05664,
Zebrafish:0.56540):0.02478,Hedgehog:0.22128):0.01141,...

Zebrafish should be the outgroup, but its branch of 0.56540, against 0.11 to 0.22 for every other tip, draws it to the next longest (long-branch attraction). This page uses the tree only to order the rows.

The alignment in the order of the ClustalW tree beside it. The three primates are adjacent rows and so are the three rodents, and Zebrafish sits next to the rodents on its long branch.

Click a node to collapse its clade into one triangle labelled with the number of tips.

Mouse, Rat and Hamster collapsed into the triangle marked 3, leaving eleven rows in the same columns.

5. Look up the domains

The CLI looks up InterPro’s precomputed Pfam matches for each accession:

react-msaview-cli interpro accessions.tsv -o family-domains.gff

InterPro release 110.0; reading precomputed pfam matches...
  [1/12] Q9C000: 9 pfam entries
  ...
12 fetched, 0 from /home/you/.cache/react-msaview-cli/interpro

For predicted proteins InterPro has never seen, react-msaview-cli interproscan submits the sequences to the EBI job queue and waits about fifteen minutes. Count what came back:

grep IPR004020 family-domains.gff | cut -f1

Human
Chimp
Rhesus
Dog
Hedgehog

Five rows out of twelve have the PYD, at residues 9-83 in each.

6. Open the three files

Load family.afa, family.nwk and family-domains.gff into the alignment, tree and optional annotation slots of the import form, as local files or URLs.

The alignment, tree and domains, with domain boxes in place of the residue colors. NACHT, the winged helix and the helical domain come first, leucine-rich repeats fall through the middle, and the two FIIND blocks and the CARD sit at the right. The PYD at the far left is drawn on five rows, and the other seven are blank there.

Settings → Annotations lists each InterPro accession in the GFF with a checkbox.

The Settings panel, one checkbox per InterPro accession with its match count. IPR004020, the pyrin domain, has 5, and IPR025307 has 24 because it matches twice in every row, as FIIND and as the UPA-FIIND block beside it.

ggmsaview takes the same three files as the alignment, tree and annotation arguments of msaview(). w opens the viewer in RStudio or a notebook, and msaview_plot() draws the view as a ggplot2 figure:

library(ggmsaview)
w <- msaview(
  msa = "https://gmod.org/JBrowseMSA/demo/data/nlrp1.aln",
  tree = "https://gmod.org/JBrowseMSA/demo/data/nlrp1.nh",
  gff = "https://gmod.org/JBrowseMSA/demo/data/nlrp1-domains.gff",
  color_scheme = "clustalx_protein_dynamic"
)
w
ggplot2::ggsave("protein-family-domains-r.png", msaview_plot(w),
  width = 12, height = 5)

The twelve rows with the GFF’s domains as filled boxes and one legend entry per domain name. The PYD at the far left covers five rows.

7. Filter to the pyrin domain

Uncheck every accession but IPR004020, and the overlay draws the PYD alone.

The overlay with only the pyrin domain turned on. The three primates and Dog have it in four adjacent rows, Hedgehog in the last row of the frame, and Cow, Pig and Horse, between them, lack it.

8. Compare residues with columns

InterPro reports NACHT at a different residue in each of the twelve rows:

grep IPR007111 family-domains.gff | cut -f1,4,5

Human	328	497
Chimp	328	497
Rhesus	332	501
Dog	299	467
Hedgehog	296	465
Cow	294	463
Pig	162	330
Horse	328	493
Mouse	134	301
Rat	176	344
Hamster	93	258
Zebrafish	258	428

Human 328 and Hamster 93 are 235 residues apart. In the viewer, Mouse, Rat, Hamster and Zebrafish begin NACHT in column 372 and the other eight rows in column 371, because the overlay maps each row’s residue coordinates through the gaps in that row before drawing.

9. Look for missing annotations

Cow, Hedgehog and Zebrafish have no leucine-rich repeats called at all:

grep IPR001611 family-domains.gff | cut -f1 | sort | uniq -c

      2 Chimp
      2 Dog
      1 Hamster
      2 Horse
      3 Human
      1 Mouse
      3 Pig
      2 Rat
      4 Rhesus

A blank region means the protein lacks the domain or its entry, often an unreviewed gene model, has no call there. At one letter per column, a row with residues under the blank differs from a row of gaps.

The left edge of the PYD block at one letter per column. The five rows with the domain are green. Cow fills all 75 of those columns with residues and has no pyrin domain called over them, and Horse fills 73 of them. Mouse, Rat and Hamster are mostly dashes there, and Hamster has 24 residues in the 75 columns.

An alignment this size opened from your own computer does not fit in the page URL, so the header says Not in the link. Put the three files on a server that sends CORS headers over HTTPS (raw.githubusercontent.com works), and name the URLs:

{
  "msaview": {
    "type": "MsaView",
    "colWidth": 0.7,
    "colorSchemeName": "clustalx_protein_dynamic",
    "msaFilehandle": { "uri": "https://example.org/family.afa" },
    "treeFilehandle": { "uri": "https://example.org/family.nwk" },
    "gffFilehandle": { "uri": "https://example.org/family-domains.gff" },
    "highlights": [{ "start": 39, "end": 113, "label": "PYD, Pfam PF02758" }]
  }
}

URL-encode that and append it to the app as #data=. The link holds the file addresses, so it works at any size. The optional highlights takes 1-based inclusive columns.

The three hosted files opened from that link, with columns 39 to 113 banded and labelled PYD across all twelve rows.

Reproduce it end to end

build_protein_family.sh runs the four commands above in order:

It fetches one sequence per accession from UniProt and names each FASTA record by its label.
ClustalW aligns the records and infers a neighbor-joining tree.
react-msaview-cli interpro writes InterPro’s precomputed Pfam matches for the accessions as a GFF, in each protein’s residue numbering.

It needs the tools under Prerequisites.

curl -O https://raw.githubusercontent.com/GMOD/JBrowseMSA/main/docs/tutorials/scripts/build_protein_family.sh
bash build_protein_family.sh

With no arguments the script writes the accession list above and builds the three files beside it. Point it at your own list for another family:

bash build_protein_family.sh my-accessions.tsv out/

References

Bateman A, et al. UniProt: the Universal Protein Knowledgebase in 2025. Nucleic Acids Research 53:D609-D617.
Blum M, et al. InterPro: the protein sequence classification resource in 2025. Nucleic Acids Research 53:D444-D456.
Larkin MA, et al. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948.
Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nature Reviews Immunology 16:407-420.