Reference APSES domains (reference species)
- Multi FASTA file of APSES domains in six fungal reference species.
This page collects APSES domain sequences from six fungal species that are used as reference species for the course. The species are:
- Aspergillus nidulans (ASPNI)
- Candida albicans (CANAL)
- Neurospora crassa (NEUCR)
- Saccharomyces cerevisiae (SACCE)
- Schizosaccharomyces pombe (SCHPO)
- Ustilago maydis (USTMA)
Executing the PSI-BLAST search
Defining the APSES Domain sequence
- Navigate to the NCBI BLAST page, accessed protein BLAST;
- Follow the link to protein BLAST and enter the yeast Mbp1 refseq ID NP_010227 into the input form;
- Select the PHI-BLAST algorithm to search for domains in the sequence and Run BLAST;
- Click on the graphical summary of the result to access the CDD conserved domains report for the sequence;
- Click on the (+) sign next to the link to KilA-N(pfam 04383) domain to display the query/profile alignment. This is what it looks like:
10 20 30 40 50 60 70 80
....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 6320147 19 IHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQ---------------GGFGKYQGTWVPLNIA 83
Cdd:pfam04383 3 YNDFEIIIRRDKDGYINATKLCKAAGAKGKRFRNWLRLESTKELIEELSkennpdkliiienrkGKGGRLQGTYVHPDLA 82
90
....*....|....
gi 6320147 84 KQLA----EKFSVY 93
Cdd:pfam04383 83 LAIAswisPEFALK 96
|
This gives us the following APSES domain sequence:
>Yeast Mbp1 APSES domain (AA 19..93 of NP_010227) IHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQG GFGKYQGTWVPLNIAKQLAEKFSVY
Searching for APSES domains
A PSI-BLAST search was executed, searching in the refseq subset of the NCBI protein database and restricting the species to the six fungal reference species plu Escherichia coli. The latter was chosen to retrieve the KilA-N domain sequence which we need as an outgroup for phylogenetic analysis.
The search converged after 5 iterations in which matches of less than 80% of the query length were manually removed, even if they had low E-values. Also, care was taken not to include false positives and thus to avoid profile corruption, and hits with E > 10-4 were also removed. The check-boxes next to the alignments were used to select sequences with > 80% coverage to the query and only the highest-scoring KilA-N domain protein was kept. Clicking on Get selected sequences created a results page of 27 sequences. These were then displayed in a FASTA(text) format and their headers were slightly edited to create a dataset of Reference APSES full length proteins.
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. To generate the multi-FASTA file of APSES domains, we could have simply adited the full length proteins manually. But there is a simpler way to achieve this. 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 a number of options to format the BLAST result page: they are presented from alink 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 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 identitites: This now looks like a multiple sequence alignment (in fact it is one - all sequences aligned to the profile).
- hit-table: this gives only the numerical parameters describing the quality of the matches.
When we select the flat-query anchored with/without identitites option, it is reasonably straightforward to obtain the aligned sequences, copy and paste them into a Word document and convert that into a multi-FASTA format with a few Edit > Replace commands.
Renaming sequences
To make the interpretation of alignments and gene trees easier, all yeast sequences were labelled with their gene name (e.g. Sok2_SACCE). Sequences that are presumed to be functionally equivalent orthologues to Mbp1 were identified through the Reciprocal Best Match criterion and labeled as Mbp1_NNNNN. All other sequences were named APS1_, APS2_, APS3_ ... - as required. (e.g. APS1_USTMA). Note that such relabeling sequences does not change the data or its interpretation, it is just helpful.
The final 27 APSES domain reference sequences
>KILA_ESCCO ZP_07189117 KilA-N domain protein IDGEIIHLRAKDGYINATSMCRTAGKLLSDYTRLKTTQEFFDELSRDMGIPISELIQSFKGGRPENQGTW VHPDIAINLAQ >MBP1_SACCE NP_010227 Mbp1 IHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKYQGTWVPLNIAKQLAE KFSVY >APS1_USTMA XP_762343 UM06196 IINNVAVMRRRSDDWLNATQILKVVGLDKPQRTRVLEREIQKGIHEKVQGGYGKYQGTWIPLDVAIELAE RYNI >APS1_NEUCR XP_962967 NCU07587 VNNVAVMRRQKDGWVNATQILKVANIDKGRRTKILEKEIQIGEHEKVQGGYGKYQGTWIPFERGLEVCRQ YGV >APS2_NEUCR XP_955821 NCU07246 VMRRRHDDWVNATHILKAAGFDKPARTRILEREVQKDTHEKIQGGYGRYQGTWIPLEQAEALARRNNIY >APS1_CANAL XP_712970 potential DNA binding component of SBF MMNESSIMRRCKDDWVNATQILKCCNFPKAKRTKILEKGVQQGLHEKVQGGFGRFQGTWIPLEDARKLAK TYGV >APS1_ASPNI XP_660758.1 AN3154 IGTDSVMRRRSDDWINATHILKVAGFDKPARTRILEREVQKGVHEKVQGGYGKYQGTWIPLQEGRQLAER NNI >APS1_SCHPO NP_595496 MBF transcription factor complex subunit Res1 INGFPLMKRCHDNWLNATQILKIAELDKPRRTRILEKFAQKGLHEKIQGGCGKYQGTWVPSERAVELAHE YNVF >APS2_SCHPO NP_593032 MBF transcription factor complex subunit Res2 IKGVSVMRRRRDSWLNATQILKVADFDKPQRTRVLERQVQIGAHEKVQGGYGKYQGTWVPFQRGVDLATK YKV >APS2_CANAL XP_723071 potential DNA binding component of MBF VTSEGPIMRRKKDSWINATHILKIAKFPKAKRTRILEKDVQTGIHEKVQGGYGKYQGTYVPLDLGAAIAR NFGVY >APS2_ASPNI XP_664319 hypothetical protein AN6715 VNGVAVMKRRSDGWLNATQILKVAGVVKARRTKTLEKEIAAGEHEKVQGGYGKYQGTWVNYQRGVELCRE YHV >APS2_USTMA XP_761485 UM05338 VRGIAVMRRRGDGWLNATQILKIAGIEKTRRTKILEKSILTGEHEKIQGGYGKFQGTWIPLQRAQQVAAE YNV >SWI4_SACCE NP_011036 Swi4p TKIVMRRTKDDWINITQVFKIAQFSKTKRTKILEKESNDMQHEKVQGGYGRFQGTWIPLDSAKFLVNKYE I >APS3_SCHPO NP_596132 MBF transcription factor complex subunit Cdc10 GDNVALRRCPDSYFNISQILRLAGTSSSENAKELDDIIESGDYENVDSKHPQIDGVWVPYDRAISIAKR YGVY >APS3_CANAL XP_714237 potential DNA binding regulator of filamentous growth NNVSVVRRADNNMINGTKLLNVAQMTRGRRDGILKSEKVRHVVKIGSMHLKGVWIPFERALAMAQREQI >SOK2_SACCE NP_013729 Sok2p NGISVVRRADNDMVNGTKLLNVTKMTRGRRDGILKAEKIRHVVKIGSMHLKGVWIPFERALAIAQREKI >APS3_ASPNI XP_663440 STUA CELL PATTERN FORMATION-ASSOCIATED PROTEIN GVCVARREDNGMINGTKLLNVAGMTRGRRDGILKSEKVRNVVKIGPMHLKGVWIPFDRALEFANKEKI >PHD1_SACCE NP_012881 Phd1p NGISVVRRADNNMINGTKLLNVTKMTRGRRDGILRSEKVREVVKIGSMHLKGVWIPFERAYILAQREQI >APS4_CANAL XP_710918 CaO19.5210 LNNHWVIWDYETGWVHLTGIWKASLTIDGSNVSPSHLKADIVKLLESTPKEYQQYIKRIRGGFLKIQGTW LPYKLCKILARRFCYY >APS3_NEUCR XP_960837 NCU01414 GICVARREDNAMINGTKLLNVAGMTRGRRDGILKSEKVRHVVKIGPMHLKGVWIPFERALDFANKEKI >APS5_CANAL XP_711513 potential DNA binding protein NILVSRREDTNYINGTKLLNVIGMTRGKRDGILKTEKIKNVVKVGSMNLKGVWIPFDRAYEIARNEGV >APS4_ASPNI XP_663009 AN5405 TVMWDYNIGLVRTTHLFKCNDYSKTTPAKMLNQNPGLRDICHSITGGALAAQGYWMPYEAAKAIAATFC >APS3_USTMA XP_760925 UM04778 VRGHTMMIDVDTSFVRFTSITQALGKNKVNFGRLVKTCPALDPHITKLKGGYLSIQGTWLPFDLAKELSR R >APS4_SCHPO NP_596166 HFLMRMAKDSSISATSMFRSAFPKATQEEEDLEMRWIRDNLNPIEDKRVAGLWVPPADALALAKDYSM >APS6_CANAL XP_723412 potential transcriptional co-activator HGEIIVLRRVQDSFVNVTQLFQILIKLEVLPTSQVDNYFDNEILSNLKYFGSSSNTPQYLDLRKHQNIYL QGIWIPYDKAVNLALKFDIY >APS4_NEUCR XP_962267 NCU06560 FLMRRSQDGYISATGMFKATFPYASQEEEEAERKYIKSIPTTSSEETAGNVWIPPEQALILAEEYQI >APS5_ASPNI XP_657766 AN0162 TYFLMRRSKDGYVSATGMFKIAFPWAKLEEERSEREYLKTRPETSEDEIAGNVWISPVLALELAAEYKMY
