Reference APSES domains (yeast)

Multi FASTA file of all saccharomyces cerevisiae APSES domains.

Executing the PSI-BLAST search

The starting point of this list is a PSI-BLAST search with one known APSES domain sequence. This query sequence - the Mbp1 APSES domain - was defined as follows, based on Pfam profile 02292: APSES.

>Yeast Mbp1 APSES domain (AA 24..102 of NP_010227)
SIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKY
QGTWVPLNIAKQLAEKFSVYDQLKPLFDF

Even though we are only interested in yeast genes, including other sequences into the PSI-BLAST search will allow us to identify distant homologues as well. A PSI-BLAST search was executed, searching in the refseq subset of GenPept and selecting an Organism restriction to use only Fungi (taxid: 4751). The default parameters for PSI-BLAST were used, except for using the BLOSUM45 matrix and an E-value threshold of 0.1, not 10.

The search converged after 6 iterations, i.e. in the 6th iteration PSI-BLAST found no additional new hits above the inclusion threshold E-value of 0.005. Clicking on the Taxonomy reports link lists all hits sorted by the species they originate from. Clicking on the Saccharomyces cerevisiae link identifies the yeast genes that were found:

 Saccharomyces cerevisiae S288c [ascomycetes] taxid 559292
ref|NP_010227.1| Mbp1p [Saccharomyces cerevisiae S288c]           126  1e-37
ref|NP_011036.1| Swi4p [Saccharomyces cerevisiae S288c]           103  9e-30
ref|NP_013729.1| Sok2p [Saccharomyces cerevisiae S288c]            98  9e-28
ref|NP_012881.1| Phd1p [Saccharomyces cerevisiae S288c]            94  2e-27
ref|NP_012165.1| Xbp1p [Saccharomyces cerevisiae S288c]            55  1e-12

One of these (Xbp1) is only a partial match with an alignment length of 55 amino acids. There is a somewhat complicated story here, therefore, for the purposes of the course I have removed Xbp1 from consideration as an APSES transcription factor. We will work from the four yeast gene families Mbp1, Swi4, Sok2, and Phd1.

Constructing the multi-FASTA file

A multi-FASTA file is the default input format for many MSA programs, it is simply a file that contains more than one FASTA formatted sequence.

The PSI-BLAST search has already defined the sequences from each source protein that are similar to the APSES search profile. We only need to extract them in a convenient way from the search results. NCBI offers options to format the result page: they are presented from a link at the top of the BLAST results page: " Reformat these Results": the principal options for the format are:

• Pairwise: the default
• Pairwise with identities: showing only differences to the query sequence
• query anchored with/without letters for identities: looks something like a multiple sequence alignment, hyphens for gaps, insertions relative to the query are displayed below the sequence
• flat-query anchored with/without letters for identities: This now looks like a multiple sequence alignment (in fact it is one - all sequences aligned to the profile).

I have selected the flat-query anchored with letters for identities option and restricted the output to saccharomyces cerevisiae sequences:

Query      1    SIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKYQGTWVPLNIA  60
NP_010227  24   SIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKYQGTWVPLNIA  83
NP_011036  60    VMRRTKDDWINITQVFKIAQFSKTKRTKILEKESNDMQHEKVQGGYGRFQGTWIPLDSA  118
NP_013729  436  SVVRRADNDMVNGTKLLNVTKMTRGRRDGILKAEKIRHV---VKIGSMHLKGVWIPFERA  492
NP_012881  208  SVVRRADNNMINGTKLLNVTKMTRGRRDGILRSEKVREV---VKIGSMHLKGVWIPFERA  264
NP_012165  343                                       NFTKRIRGGYIKIQGTWLPMEIS  365

Query      61   KQLAEKFS--VYD-QLKPLFDF  79
NP_010227  84   KQLAEKFS--VYD-QLKPLFDF  102
NP_011036  119  KFLVNKYE--IIDPVVNSILTF  138
NP_013729  493  LAIAQREK--IAD-YLYPLF    509
NP_012881  265  YILAQREQ--ILD-HLYPLF    281
NP_012165  366  RLLCLRFCFPIRY-FLVPIFG   385

I can then simply copy the alignment, remove hyphens and create FASTA headers to which I have manually added some useful information. The "Query" itself (being identical to the original Mbp1 protein) and the Xbp1 partial match are not included.

Yeast APSES domains

This is the final yeast APSES domain reference sequence set in multi-FASTA format.

>Mbp1_SACCE (79  ids)  NP_010227    (024..102)
SIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKYQGTWVPLNIAKQLAEKFSVYDQLKPLFDF
>Sok2_SACCE (74  ids)  NP_013729     (436..509)
SVVRRADNDMVNGTKLLNVTKMTRGRRDGILKAEKIRHVVKIGSMHLKGVWIPFERALAIAQREKIADYLYPLF
>Phd1_SACCE (74  ids)  NP_012881    (208..281)
SVVRRADNNMINGTKLLNVTKMTRGRRDGILRSEKVREVVKIGSMHLKGVWIPFERAYILAQREQILDHLYPLF
>Swi4_SACCE (79  ids)  NP_011036    (060..138)
VMRRTKDDWINITQVFKIAQFSKTKRTKILEKESNDMQHEKVQGGYGRFQGTWIPLDSAKFLVNKYEIIDPVVNSILTF