BZIP study

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Contents

Identification

Accession IDs for bZIP transcription factor domain:

  1. Pfam: PF00170
  2. SMART: SM00338
  3. Interpro: IPR004827
  4. PubMed (NCBI): PUBMED:7780801


Pfam-based approach

HMM domain clans

Protein domain information obtained from Pfam database.

Regarded as the main domain for bZIP transcription factor. Clearly distinguishes basic region and leucine zipper.

Used for further analysis.

Conserved basic region, weaker leucine zipper.

  • bZIP_Maf (PF03131)

No distinct basic region. No clear leucine zipper interface.

Downloading and generating HMM consensus sequence file

WD: 63:/data6/chojam96/bZIP/identification
  • Download Stockholm alignment file from Pfam bZIP_1 > Alignment > Download options > Seed

Seed: the curated alignment from which the HMM for the family is built.

  • Use 'hmmbuild' command from HMMER software to convert Stockholm alignment into a profile HMM

After gunzipping, use following command.

hmmbuild [options] hmmfile alignfile

hmmbuild reads a multiple sequence alignment file alignfile , builds a new profile HMM, and saves the HMM in hmmfile.

For following commands for HMMER, refer to this page.

hmmbuild PF00170.hmm PF00170.seed

hmmscanning the protein sequence

  • Use 'hmmpress' command for preparing HMM database
hmmpress [options] hmmfile

This produces four preparation files.

  • Run 'hmmscan' command against Vigna radiata pepetide sequence fasta file
hmmscan [options] hmmdb seqfile
hmmscan --tblout PF00170.out PF00170.hmm Vradi_ver6.fa.cds.primary.fasta.pepshorten.fa

No primary options used. Peptide file used for hmmscanning had only main transcripts(*.1, n=22427).

PF00170.out (result file of hmmscan) contained 147 nonredundant genes.

Out of 147 genes, 144 genes had complete peptide sequence, marked by termination codon sign (*).

  • Get corresponding protein sequences
python getgenes.py PF00170.out Vradi_ver6.fa.cds.primary.fasta.pepshorten.fa bZIP.primary.fa

Confirming domain presence

Trickest part in identification of bZIP genes - the best way is to pick and choose gene by gene, with measures.

1. Align peptide sequence file (bZIP.primary.fa, n=147) by 'MUSCLE' to manually see the alignment

muscle -in bZIP.primary.fa -out bZIP.primary.aligned.fa

Toggled sequence at 50% level to check conserved domains, but no consensus found.

2. Use 'interproscan' or 'NCBI CD search' for domain detection