Difference between revisions of "Reference APSES domains (reference species)"
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__NOTOC__ | __NOTOC__ | ||
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| + | <div class="alert"> | ||
| + | The species used on this page are not the current set of [[Reference_species_for_fungi|reference species]]. Proceed with caution. | ||
| + | </div> | ||
<section begin=contents_summary /> | <section begin=contents_summary /> | ||
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| − | * see also: [[ | + | * see also: [[Reference APSES proteins (reference species)]] |
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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 plus ''Escherichia coli''. The latter was chosen to retrieve the KilA-N domain sequence which we need as an outgroup for phylogenetic analysis. | 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 plus ''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 | + | 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 the dataset [[Reference APSES proteins (reference species)]]. |
===Constructing the multi-FASTA file=== | ===Constructing the multi-FASTA file=== | ||
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Here is a sample set of the APSES domain sequences to illustrate the '''phylip''' format. Sequences were aligned with MAFFT and edited in JALVIEW to remove gapped regions and frayed termini. The FASTA sequences were converted with [http://www-bimas.cit.nih.gov/molbio/readseq/ the Readseq server]. | Here is a sample set of the APSES domain sequences to illustrate the '''phylip''' format. Sequences were aligned with MAFFT and edited in JALVIEW to remove gapped regions and frayed termini. The FASTA sequences were converted with [http://www-bimas.cit.nih.gov/molbio/readseq/ the Readseq server]. | ||
| − | < | + | <pre> |
27 78 | 27 78 | ||
KILA_ESCCO DGEIIHLRAK DGYINATSMC RT-A-GKLLS DYTRLKLSRD M-GIPIS-IQ | KILA_ESCCO DGEIIHLRAK DGYINATSMC RT-A-GKLLS DYTRLKLSRD M-GIPIS-IQ | ||
| Line 251: | Line 256: | ||
--TSSEETAG NVWIPPEQAL ILAEEYQI | --TSSEETAG NVWIPPEQAL ILAEEYQI | ||
| − | </ | + | </pre> |
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* Paste the following organism restrictions into the '''Entrez query''' field. This includes all fungi we have worked with in the course, as well as ''Escherichia coli'' (for the KilA-N domain): | * Paste the following organism restrictions into the '''Entrez query''' field. This includes all fungi we have worked with in the course, as well as ''Escherichia coli'' (for the KilA-N domain): | ||
| − | < | + | <pre> |
Ajellomyces dermatitidis [ORGN] | Ajellomyces dermatitidis [ORGN] | ||
OR Arthroderma benhamiae [ORGN] | OR Arthroderma benhamiae [ORGN] | ||
| Line 331: | Line 336: | ||
OR Zymoseptoria tritici [ORGN] | OR Zymoseptoria tritici [ORGN] | ||
OR Escherichia coli [ORGN] | OR Escherichia coli [ORGN] | ||
| − | </ | + | </pre> |
* Select '''PSI-BLAST''' as the algorithm. | * Select '''PSI-BLAST''' as the algorithm. | ||
* '''BLAST''' this. | * '''BLAST''' this. | ||
| Line 381: | Line 386: | ||
====Processing the PSI-BLAST results==== | ====Processing the PSI-BLAST results==== | ||
* We need to collapse the separate aligned sections, remove the profusion of gap characters, and replace the semantically meaningless GI numbers with something that we can use for interpreting alignments and trees. I could do this by hand for the ~300 sequences in about 2 hours. I chose to write some Perl code instead. It works on the copied alignments, the headers, and the RBM annotations. | * We need to collapse the separate aligned sections, remove the profusion of gap characters, and replace the semantically meaningless GI numbers with something that we can use for interpreting alignments and trees. I could do this by hand for the ~300 sequences in about 2 hours. I chose to write some Perl code instead. It works on the copied alignments, the headers, and the RBM annotations. | ||
| − | < | + | <pre> |
#!/usr/bin/perl | #!/usr/bin/perl | ||
# ProcessPSI-BLAST.pl | # ProcessPSI-BLAST.pl | ||
| Line 491: | Line 496: | ||
exit(); | exit(); | ||
| − | </ | + | </pre> |
====Alignment==== | ====Alignment==== | ||
* The alignment was done at the EBI using MAFFT and written using FASTA output format. | * The alignment was done at the EBI using MAFFT and written using FASTA output format. | ||
| − | < | + | <pre> |
>Mbp1_USTMA XP_762343 | >Mbp1_USTMA XP_762343 | ||
--------IIN-NVA-VMRRRSDDWLN--------------------------------- | --------IIN-NVA-VMRRRSDDWLN--------------------------------- | ||
| Line 1,916: | Line 1,921: | ||
ELIQ-----------SFKGG----------------RP---ENQ-------GTW------ | ELIQ-----------SFKGG----------------RP---ENQ-------GTW------ | ||
-------------VHPDIAINLAQ----- | -------------VHPDIAINLAQ----- | ||
| − | </ | + | </pre> |
--> | --> | ||
Latest revision as of 06:51, 26 September 2020
Reference APSES domains
The species used on this page are not the current set of reference species. Proceed with caution.
Sequences of APSES domains in the fungal reference species - domain definition, PSI-BLAST search, and header editing.
The APSES domain proteins were determined with a PSI-BLAST search in the refseq database, using 1BM8_A as the search sequence, and restricting the search to the Reference species for fungi.
Executing the PSI-BLAST search
Defining the APSES Domain sequence
- The APSES domain "proper"
- 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 plus 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 the dataset Reference APSES proteins (reference species).
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 edited 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 a link at the top of the BLAST results page: "Formatting options": 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 letters for 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 Saccharomyces cerevisiaea 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 (RBM) criterion and labeled as Mbp1_NNNNN. All other sequences were named APS1_, APS2_, APS3_ ... - as required. (e.g. APS1_USTMA). There is no further significance in the numbers, i.e. APS1_USTMA is not necessarily an RBM to APS1_SCHPO. Note that such relabeling of sequences does not change the data or its interpretation, it is just helpful to interpret the tree.
The final 27 APSES domain reference sequences
>KILA_ESCCO ZP_07189117 KilA-N domain protein IDGEIIHLRAKDGYINATSMCRTAGKLLSDYTRLKTTQEFFDELSRDMGIPISELIQSFKGGRPENQGTW VHPDIAINLAQ >MBP1_SACCE NP_010227 Mbp1 IHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKYQGTWVPLNIAKQLAE KFSVY >MBP1_USTMA XP_762343 UM06196 IINNVAVMRRRSDDWLNATQILKVVGLDKPQRTRVLEREIQKGIHEKVQGGYGKYQGTWIPLDVAIELAE RYNI >MBP1_NEUCR XP_955821 NCU07246 VMRRRHDDWVNATHILKAAGFDKPARTRILEREVQKDTHEKIQGGYGRYQGTWIPLEQAEALARRNNIY >MBP1_ASPNI XP_660758.1 AN3154 IGTDSVMRRRSDDWINATHILKVAGFDKPARTRILEREVQKGVHEKVQGGYGKYQGTWIPLQEGRQLAER NNI >MBP1_SCHPO NP_593032 MBF transcription factor complex subunit Res2 IKGVSVMRRRRDSWLNATQILKVADFDKPQRTRVLERQVQIGAHEKVQGGYGKYQGTWVPFQRGVDLATK YKV >MBP1_CANAL XP_723071 potential DNA binding component of MBF VTSEGPIMRRKKDSWINATHILKIAKFPKAKRTRILEKDVQTGIHEKVQGGYGKYQGTYVPLDLGAAIAR NFGVY >APS1_NEUCR XP_962967 NCU07587 VNNVAVMRRQKDGWVNATQILKVANIDKGRRTKILEKEIQIGEHEKVQGGYGKYQGTWIPFERGLEVCRQ YGV >APS1_CANAL XP_712970 potential DNA binding component of SBF MMNESSIMRRCKDDWVNATQILKCCNFPKAKRTKILEKGVQQGLHEKVQGGFGRFQGTWIPLEDARKLAK TYGV >APS1_SCHPO NP_595496 MBF transcription factor complex subunit Res1 INGFPLMKRCHDNWLNATQILKIAELDKPRRTRILEKFAQKGLHEKIQGGCGKYQGTWVPSERAVELAHE YNVF >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
Mbp1 orthologue reference alignment
This is a reference alignment of the APSES domains of those proteins that fulfilled the Reciprocal Best Match criterion with yeast Mbp1.
CLUSTAL format alignment by MAFFT L-INS-1 (v6.850b) MBP1_SACCE IHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKYQGTWVPLNIAKQLAEKFSVY MBP1_CANAL VTSEGPIMRRKKDSWINATHILKIAKFPKAKRTRILEKDVQTGIHEKVQGGYGKYQGTYVPLDLGAAIARNFGVY MBP1_USTMA IINNVAVMRRRSDDWLNATQILKVVGLDKPQRTRVLEREIQKGIHEKVQGGYGKYQGTWIPLDVAIELAERYNI- MBP1_NEUCR ------VMRRRHDDWVNATHILKAAGFDKPARTRILEREVQKDTHEKIQGGYGRYQGTWIPLEQAEALARRNNIY MBP1_ASPNI -IGTDSVMRRRSDDWINATHILKVAGFDKPARTRILEREVQKGVHEKVQGGYGKYQGTWIPLQEGRQLAERNNI- MBP1_SCHPO -IKGVSVMRRRRDSWLNATQILKVADFDKPQRTRVLERQVQIGAHEKVQGGYGKYQGTWVPFQRGVDLATKYKV-
Sample Phylip format
Here is a sample set of the APSES domain sequences to illustrate the phylip format. Sequences were aligned with MAFFT and edited in JALVIEW to remove gapped regions and frayed termini. The FASTA sequences were converted with the Readseq server.
27 78
KILA_ESCCO DGEIIHLRAK DGYINATSMC RT-A-GKLLS DYTRLKLSRD M-GIPIS-IQ
MBP1_SACCE STGSIMKRKK DDWVNATHIL KA-A-NFAKA KRTRI-LEKE V-LKETH--E
MBP1_USTMA NNVAVMRRRS DDWLNATQIL KV-V-GLDKP QRTRV-LERE I-QKGIH--E
MBP1_NEUCR ----VMRRRH DDWVNATHIL KA-A-GFDKP ARTRI-LERE V-QKDTH--E
MBP1_ASPNI GTDSVMRRRS DDWINATHIL KV-A-GFDKP ARTRI-LERE V-QKGVH--E
MBP1_SCHPO KGVSVMRRRR DSWLNATQIL KV-A-DFDKP QRTRV-LERQ V-QIGAH--E
MBP1_CANAL SEGPIMRRKK DSWINATHIL KI-A-KFPKA KRTRI-LEKD V-QTGIH--E
APS1_NEUCR NNVAVMRRQK DGWVNATQIL KV-A-NIDKG RRTKI-LEKE I-QIGEH--E
APS1_CANAL NESSIMRRCK DDWVNATQIL KC-C-NFPKA KRTKI-LEKG V-QQGLH--E
APS1_SCHPO NGFPLMKRCH DNWLNATQIL KI-A-ELDKP RRTRI-LEKF A-QKGLH--E
APS2_ASPNI NGVAVMKRRS DGWLNATQIL KV-A-GVVKA RRTKT-LEKE I-AAGEH--E
APS2_USTMA RGIAVMRRRG DGWLNATQIL KI-A-GIEKT RRTKI-LEKS I-LTGEH--E
SWI4_SACCE -TKIVMRRTK DDWINITQVF KI-A-QFSKT KRTKI-LEKE S-NDMQH--E
APS3_SCHPO GDNVALRRCP DSYFNISQIL RL-A-GTSSS ENAKE-LDDI I-ESGDY--E
APS3_CANAL NNVSVVRRAD NNMINGTKLL NV-A-QMTRG RRDGI-LKSE ----KVR--H
SOK2_SACCE NGISVVRRAD NDMVNGTKLL NV-T-KMTRG RRDGI-LKAE ----KIR--H
APS3_ASPNI -GVCVARRED NGMINGTKLL NV-A-GMTRG RRDGI-LKSE ----KVR--N
PHD1_SACCE NGISVVRRAD NNMINGTKLL NV-T-KMTRG RRDGI-LRSE ----KVR--E
APS4_CANAL NNHWVIWDYE TGWVHLTGIW KA-SLSHLKA DIVKL-LEST PKEYQQY-IK
APS3_NEUCR -GICVARRED NAMINGTKLL NV-A-GMTRG RRDGI-LKSE ----KVR--H
APS5_CANAL -NILVSRRED TNYINGTKLL NV-I-GMTRG KRDGI-LKTE ----KIK--N
APS4_ASPNI ---TVMWDYN IGLVRTTHLF KC-N-DYSKT TPAKM-LNQN PGLRDIC--H
APS3_USTMA RGHTMMIDVD TSFVRFTSIT QA-L-GKNKV NFGRL-VKTC P-ALDPH-IT
APS4_SCHPO --HFLMRMAK DSSISATSMF RS-A-FPKAT QEEED-LEMR WIRDNLN---
APS6_CANAL GEIIVLRRVQ DSFVNVTQLF QILE-VLPTS QVDNY-FDNE I-LSNLKYLR
APS4_NEUCR ---FLMRRSQ DGYISATGMF KA-T-FPYAS QEEEE-AERK YIKSIPT---
APS5_ASPNI -TYFLMRRSK DGYVSATGMF KI-A-FPWAK LEEER-SERE YLKTRPE---
SFKGGRPENQ GTWVHPDIAI NLAQ----
KVQGGFGKYQ GTWVPLNIAK QLAEKFSV
KVQGGYGKYQ GTWIPLDVAI ELAERYNI
KIQGGYGRYQ GTWIPLEQAE ALARRNNI
KVQGGYGKYQ GTWIPLQEGR QLAERNNI
KVQGGYGKYQ GTWVPFQRGV DLATKYKV
KVQGGYGKYQ GTYVPLDLGA AIARNFGV
KVQGGYGKYQ GTWIPFERGL EVCRQYGV
KVQGGFGRFQ GTWIPLEDAR KLAKTYGV
KIQGGCGKYQ GTWVPSERAV ELAHEYNV
KVQGGYGKYQ GTWVNYQRGV ELCREYHV
KIQGGYGKFQ GTWIPLQRAQ QVAAEYNV
KVQGGYGRFQ GTWIPLDSAK FLVNKYEI
NVDSKHPQID GVWVPYDRAI SIAKRYGV
VVKIGSMHLK GVWIPFERAL AMAQREQI
VVKIGSMHLK GVWIPFERAL AIAQREKI
VVKIGPMHLK GVWIPFDRAL EFANKEKI
VVKIGSMHLK GVWIPFERAY ILAQREQI
RIRGGFLKIQ GTWLPYKLCK ILARRFCY
VVKIGPMHLK GVWIPFERAL DFANKEKI
VVKVGSMNLK GVWIPFDRAY EIARNEGV
SITGGALAAQ GYWMPYEAAK AIAATFC-
KLKGGYLSIQ GTWLPFDLAK ELSRR---
--PIEDKRVA GLWVPPADAL ALAKDYSM
KHQNIY--LQ GIWIPYDKAV NLALKFDI
--TSSEETAG NVWIPPEQAL ILAEEYQI
