View source for Reference APSES domains (reference species)

__NOTOC__

;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 [http://www.ncbi.nlm.nih.gov/blast 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 [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=10&seltype=2&uid=190963 KilA-N(pfam 04383)] domain to display the query/profile alignment. This is what it looks like:

<table>
<tr><td>
 <font color=#700777>                          10        20        30        40        50        60        70        80</font>
 <font color=#700777>                  ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|</font>
 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&doptcmdl=GenPept&db=Protein&term=6320147 gi 6320147]    <font color=#229922> 19 </font><font color=#2233CC>IHSTGS</font><font color=#FF4466>I</font><font color=#2233CC>MK</font><font color=#FF4466>R</font><font color=#2233CC>K</font><font color=#FF4466>KD</font><font color=#2233CC>DWV</font><font color=#FF4466>NAT</font><font color=#2233CC>HIL</font><font color=#FF4466>KAA</font><font color=#2233CC>NFAKAKRTRI</font><font color=#FF4466>L</font><font color=#2233CC>EK</font><font color=#FF4466>E</font><font color=#2233CC>VL</font><font color=#FF4466>KE</font><font color=#2233CC>TH</font><font color=#FF4466>E</font><font color=#2233CC>KVQ</font><font color=#888888>---------------</font><font color=#FF4466>G</font><font color=#2233CC>GF</font><font color=#FF4466>G</font><font color=#2233CC>KY</font><font color=#FF4466>QGT</font><font color=#2233CC>W</font><font color=#FF4466>V</font><font color=#2233CC>PLNI</font><font color=#FF4466>A</font> <font color=#229922>83</font>
 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&doptcmdl=GenPept&db=cdd&term=pfam04383 Cdd:pfam04383] <font color=#229922>  3 </font><font color=#2233CC>YNDFEI</font><font color=#FF4466>I</font><font color=#2233CC>IR</font><font color=#FF4466>R</font><font color=#2233CC>D</font><font color=#FF4466>KD</font><font color=#2233CC>GYI</font><font color=#FF4466>NAT</font><font color=#2233CC>KLC</font><font color=#FF4466>KAA</font><font color=#2233CC>GAKGKRFRNW</font><font color=#FF4466>L</font><font color=#2233CC>RL</font><font color=#FF4466>E</font><font color=#2233CC>ST</font><font color=#FF4466>KE</font><font color=#2233CC>LI</font><font color=#FF4466>E</font><font color=#2233CC>ELS</font><font color=#888888>kennpdkliiienrk</font><font color=#FF4466>G</font><font color=#2233CC>KG</font><font color=#FF4466>G</font><font color=#2233CC>RL</font><font color=#FF4466>QGT</font><font color=#2233CC>Y</font><font color=#FF4466>V</font><font color=#2233CC>HPDL</font><font color=#FF4466>A</font> <font color=#229922>82</font>
 
 
 <font color=#700777>                          90</font>
 <font color=#700777>                  ....*....|....</font>
 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&doptcmdl=GenPept&db=Protein&term=6320147 gi 6320147]    <font color=#229922> 84 </font><font color=#2233CC>KQL</font><font color=#FF4466>A</font><font color=#888888>----</font><font color=#2233CC>EK</font><font color=#FF4466>F</font><font color=#2233CC>SVY</font> <font color=#229922>93</font>
 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&doptcmdl=GenPept&db=cdd&term=pfam04383 Cdd:pfam04383] <font color=#229922> 83 </font><font color=#2233CC>LAI</font><font color=#FF4466>A</font><font color=#888888>swis</font><font color=#2233CC>PE</font><font color=#FF4466>F</font><font color=#2233CC>ALK</font> <font color=#229922>96</font>
</td></tr>
</table>

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<sup>-4</sup> 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. <code>Sok2_SACCE</code>). Sequences that are presumed to be functionally equivalent orthologues to Mbp1 were identified through the ''Reciprocal Best Match'' criterion and labeled as <code>Mbp1_NNNNN</code>. All other sequences were named APS1_, APS2_, APS3_ ... - as required.   (e.g. <code>APS1_USTMA</code>). 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