Reference APSES domains (reference species)

Multi FASTA file of all APSES domains in fungal proteins.

Executing the PSI-BLAST search

The starting point of this list is a BLAST search with one known APSES domain sequence. This query sequence - the Mbp1 APSES domain - was defined as follows

>Yeast Mbp1 APSES domain (AA 24..107 of NP_010227)
SIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKY
QGTWVPLNIAKQLAEKFSVYDQLKPLFDFTQTDG

A PSI-BLAST search was executed, searching in the nr subset of GenPept without further restrictions. The default parameters for PSI-BLAST were used, except for using the BLOSUM45 matrix and reducing the Evalue to 1.0 from 10.0.

The search converged after 6 iterations, i.e. PSI-BLAST had found no additional new hits above the inclusion threshold E-value of 0.005. 169 sequences were found and contributed to the profile. However, some of these sequences are redundant, i.e. they are matches to the same amiono acid sequence in different database entries, and some of these sequences are from organisnms other than the ones we are considering in the assignment. Even if these latter sequences are removed, it was appropriate to keep them included initially: they contribute to the information in the PSI-BLAST search profile and improve the sensitivity and specificity of the search.

It would certainly not be impossible - albeit somewhat tedious - to manually edit the list of proteins by checking/unchecking which hits to include. I have written a short Perl script for this task solely to be able to rename the sequences at the same time. This is not required; RefSeq / GenPept accession numbers will do just fine to name the sequences, but the final analysis is much easier to do if the sequence labels actually tell us something about the organisms they came from and which other sequence they might be similar to.

After removing redundant sequences, sequence fragments that did not span the entire Mbp1 APSES domain and sequences from fungi that are not in the list of organisms for this course, XXX sequences remain.

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The search returned 81 hits with significant e-values by the 5th iteration. 5 of these were from the organism Chaetomia globosum and were removed from the list since this is not one of the organisms we are studying. 6 hits were aligned only along a part of the APSES domain. For five of these hits, reasonable similarity to the whole APSES domain was independently verified by manually performing a Needleman-Wunsch optimal alignment with the Mbp1 APSES domain sequence. (EMBOSS NEEDLE using EBLOSUM 30, default gap parameters).

However the match to the Neurospora crassa protein XP_962373 suggested an incorrect gene model. Consider the alignment:

QUERY       1 SIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKY     50
                                          .:.:.:.||:....:.||..|.
XP_962373   1                             MLNQNPGLKDIAYSITGGAIKA     22

QUERY      51 QGTWVPLNIAKQLAEKF--SVYDQLKPLF--DFTQ---TDG              84
              ||.|.|:..||::...|  .:..:|.|||  ||..   :.|         
XP_962373  23 QGYWMPYACAKAVCATFCYQIAGALIPLFGPDFPSECISPGEPRYGIMII     72

In this situation you have to be suspicious that the gene-finder algorithm skipped a part of the N-terminus. Or, the sequence was derived from a partial m-RNA. This sequence was removed from analysis.

Further, XP_712876 and XP_712970 were found to be identical sequences from the same organism. Only one of these duplicates was kept.

This gave a total of 74 ASPES domain sequences for analysis.

A multi-FASTA file

Since we are interested in only the APSES domain, we need to display the search results in an appropriate format. If we navigate to the page from where we sent the BLAST query, we have several options to display search results:

• 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.

Using 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. Of course, the sequences for which only partial matches were found need to be completed "by hand" (from the reults of the pairwise sequence alignment described above to validate these sequences).

Renaming sequences

To support the interpretation of alignments and gene trees, the Mbp1 orthologues for all species were named accordingly (e.g. MBP1_ASPFU). All yeast genes were given the yeast-gene-name (e.g. SOK2_SACCE). All other sequences were named with the last four digits of their RefSeq ID and a five character species code according to their species (e.g. SOK2_SACCE). This is a pain to do by hand, so I wrote a little perl script to parse this information from the original BLAST report and modify the headers in the multi-FASTA file accordingly. However, note that renaming sequences does not change the data or its interpretation, it is just helpful.

The final 74 sequences

>MBP1_SACCE NP_010227 024..107
SIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKYQGTWVPLNIAKQLAEKFSVYDQLKPLFDFTQTDG
>MBP1_YARLI XP_500257 022..105
AVMRRKSDGWVNATHILKVAGFDKPQRTRILEKEVQKGVHEKVQGGYGKYQGTWVPLERAREIATLYDVDSHLAPIFNYDDEDG
>5821_NEUCR XP_955821 037..118
VMRRRHDDWVNATHILKAAGFDKPARTRILEREVQKDTHEKIQGGYGRYQGTWIPLEQAEALARRNNIYERLKPIFEFQPGN
>9090_CRYNE XP_569090 036..117
AVMRRRSDAYLNATQILKVAGFDKPQRTRVLEREVQKGEHEKVQGGYGKYQGTWIPIERGLALAKQYGVEDILRPIIDYVPT
>MBP1_ASPNI XP_660758 028..110
SVMRRRSDDWINATHILKVAGFDKPARTRILEREVQKGVHEKVQGGYGKYQGTWIPLQEGRQLAERNNILDKLLPIFDYVAGD
>MBP1_KLULA XP_454189 025..108
SIMKRKADNWVNATHILKAAKFPKAKRTRILEKEVITDTHEKVQGGFGKYQGTWIPLELASKLAEKFEVLDELKPLFDFTQQEG
>MBP1_GIBZE XP_384396 045..129
AVMRRRNDSWLNATQILKVAGVDKGKRTKILEKEIQTGEHEKVQGGYGKYQGTWIKFERGLQVCRQYGVEELLRPLLTYDMGQDG
>MBP1_ASPTE XP_001213217 028..110
SVMRRRADDWINATHILKVAGFDKPARTRILEREVQKGVHEKVQGGYGKYQGTWIPLPEGRLLAERNNIIDKLRPIFDYVAGD
>MBP1_CANAL XP_723071 026..108
IMRRKKDSWINATHILKIAKFPKAKRTRILEKDVQTGIHEKVQGGYGKYQGTYVPLDLGAAIARNFGVYDVLKPIFEFQYIEG
>MBP1_CANGL XP_445458 024..107
SIMKRKNDGWVNATHILKAANFAKAKRTRILEKEVLKEMHEKVQGGFGKYQGTWVPLNIAINLAEKFDVYQDLKPLFDFSEENG
>1770_YARLI XP_501770 036..116
AVMRRRTDSSLNATQILKVAGVEKSKRTKILEKEILTGAHEKVQGGYGKYQGTWIPYERGVDLCRQYSVYDVLQPLLAFDP
>2974_MAGGR XP_362974 121..199
VMRRRVDDWINATHILKAAGFDKPARTRILEREVQKDQHEKVQGGYGKYQGTWIPLEAGEALAHRNNIFDRLRPIFEFS
>1485_USTMA XP_761485 182..262
AVMRRRGDGWLNATQILKIAGIEKTRRTKILEKSILTGEHEKIQGGYGKFQGTWIPLQRAQQVAAEYNVSHLLQPILEFDP
>MBP1_USTMA XP_762343 026..107
AVMRRRSDDWLNATQILKVVGLDKPQRTRVLEREIQKGIHEKVQGGYGKYQGTWIPLDVAIELAERYNIQGLLQPITSYVPS
>0560_GIBZE XP_390560 040..120
VMRRRSDDWINATHILKAAGFDKPARTRILERDVQKDVHEKIQGGYGKYQGTWIPLESGQALAERHSVIDRLRPIFEYVQG
>4232_ASPFU XP_754232 001..081
MRRRGDDWINATHILKVAGFDKPARTRILEREVQKGTHEKVQGGYGKYQGTWIPLHEGRLLAERNNIIDKLRPIFDYVAGD
>MBP1_CRYNE XP_570545 133..214
SVMRRASDSWVNATQILKVAGVHKSARTKILEKEVLNGIHEKIQGGYGKYQGTWVPLDRGRDLAEQYGVGSYLSSVFDFVPS
>MBP1_NEUCR XP_962967 071..155
AVMRRQKDGWVNATQILKVANIDKGRRTKILEKEIQIGEHEKVQGGYGKYQGTWIPFERGLEVCRQYGVEELLSKLLTHNRGQEG
>MBP1_DEBHA XP_458784 027..109
IMRRKLDSWINATHILKIAKFPKAKRTRILEKDVQTGVHEKVQGGYGKYQGTYVPLDLGADIAKNFGVFDSLRPIFEFTYVEG
>2876_CANAL XP_712876 006..088
SIMRRCKDDWVNATQILKCCNFPKAKRTKILEKGVQQGLHEKVQGGFGRFQGTWIPLEDARRLAKTYGVTEELAPVLFLDFSD
>MBP1_MAGGR XP_365024 131..210
AVMKRIGDSKLNATQILKVAGVEKGKRTKILEKEIQTGEHEKVQGGYGKYQGTWIKYERALEVCRQYGVEELLRPLLEYN
>4319_ASPNI XP_664319 119..198
AVMKRRSDGWLNATQILKVAGVVKARRTKTLEKEIAAGEHEKVQGGYGKYQGTWVNYQRGVELCREYHVEELLRPLLEYD
>MBP1_ASPFU XP_748947 105..184
AVMKRRSDSWLNATQILKVAGVVKARRTKTLEKEIAAGEHEKVQGGYGKYQGTWVNYQRGVELCREYHVEELLRPLLEYD
>MBP1_SCHPO NP_593032 027..110
SVMRRRRDSWLNATQILKVADFDKPQRTRVLERQVQIGAHEKVQGGYGKYQGTWVPFQRGVDLATKYKVDGIMSPILSLDIDEG
>5548_ASPTE XP_001215548 007..086
AVMKRRSDSWLNATQILKVAGVVKARRTKTLEKEIAAGEHEKVQGGYGKYQGTWVNYQRGVDLCREYHVEELLRPLLEYD
>5496_SCHPO NP_595496 026..106
LMKRCHDNWLNATQILKIAELDKPRRTRILEKFAQKGLHEKIQGGCGKYQGTWVPSERAVELAHEYNVFDLIQPLIEYSGS
>7246_DEBHA XP_457246 028..109
IMRRCKDDWVNATQILKCCNFPKAKRTKILEKGVQQGLHEKIQGGYGRFQGTWIPLADAQRLAASYGVTPDLAPVLYLDASD
>MBP1_EREGO NP_986147 031..114
SIMKRKADDWVNATHILKAAKFAKAKRTRILEKEVIKDTHEKVQGGFGKYQGTWVPLDIARRLAQKFEVLEELRPLFDFTRRDG
>6370_EREGO NP_986370 043..124
VMRRLHDDWVNITQVFKVATFSKTQRTKILEKESADISHEKIQGGYGRFQGTWIPLDSAKGLVAKYEITDIVVLTVINFQPD
>SWI4_SACCE NP_011036 060..141
VMRRTKDDWINITQVFKIAQFSKTKRTKILEKESNDMQHEKVQGGYGRFQGTWIPLDSAKFLVNKYEIIDPVVNSILTFQFD
>4890_KLULA XP_454890 119..200
IMRRCNDNWLNITQVFKAGSFTKAQRTKILEKEANEIKHEKIQGGYGRFQGTWIPWESTKYLVEKYNINNKVVKRIVEFIPD
>4966_CANGL XP_444966 062..140
VMRRTMDDWVNVTQVFKIAQFSKTQRTKILEKESTNMKHEKVQGGYGRFQGTWVPLEAAKFMTTKYNIDNPVVNTILSF
>9785_DEBHA XP_459785 307..380
SVVRRADNNMINGTKLLNVAQMTRGRRDGILKSEKVRHVVKIGSMHLKGVWIPFERALAMAQREGIVDLLYPLF
>3009_ASPNI XP_663009 131..216
TVMWDYNIGLVRTTHLFKCNDYSKTTPAKMLNQNPGLRDICHSITGGALAAQGYWMPYEAAKAIAATFCWKIRFALTPLFGDNFPD
>SOK2_SACCE NP_013729 436..509
SVVRRADNDMVNGTKLLNVTKMTRGRRDGILKAEKIRHVVKIGSMHLKGVWIPFERALAIAQREKIADYLYPLF
>9680_CANGL XP_449680 143..216
TVVRRADNDMVNGTKLLNVTGMTRGRRDGILKNEPVRDVVKGGPMTLKGVWIPIDRARAIARQEGIEQWLYPLF
>3001_EREGO NP_983001 352..425
SVVRRADNDMINGTKLLNVAKMTRGRRDGILKAEKVRHVVKIGSMHLKGVWIPFERALALAQREKIVDMLFPLF
>4197_CANAL XP_714197 227..300
SVVRRADNNMINGTKLLNVAQMTRGRRDGILKSEKVRHVVKIGSMHLKGVWIPFERALAMAQREQIVDMLYPLF
>4237_CANAL XP_714237 228..301
SVVRRADNNMINGTKLLNVAQMTRGRRDGILKSEKVRHVVKIGSMHLKGVWIPFERALAMAQREQIVDMLYPLF
>8256_ASPTE XP_001218256 139..211
VARREDNSMINGTKLLNVAGMTRGRRDGILKSEKIRHVVKIGPMHLKGVWIPFERALEFANKEKITDLLYPLF
>3440_ASPNI XP_663440 152..224
VARREDNGMINGTKLLNVAGMTRGRRDGILKSEKVRNVVKIGPMHLKGVWIPFDRALEFANKEKITDLLYPLF
>2292_YARLI XP_502292 285..357
VARREDNDMINGTKLLNVAGMTRGRRDGILKGEKLRHVVKAGAMHLKGVWIPYDRALEFANKEKIIDLLFPLF
>1102_YARLI XP_501102 130..202
VARREDNNMINGTKLLNVVGMTRGRRDGILKTEKIRHVVKIGAMHLKGVWIPYERALAFAQRERIVDVLYPLF
>5125_ASPFU XP_755125 152..224
VARREDNHMINGTKLLNVAGMTRGRRDGILKSEKVRHVVKIGPMHLKGVWIPFERALEFANKEKITDLLYPLF
>PHD1_SACCE NP_012881 208..281
SVVRRADNNMINGTKLLNVTKMTRGRRDGILRSEKVREVVKIGSMHLKGVWIPFERAYILAQREQILDHLYPLF
>8847_CANGL XP_448847 224..297
SVVRRADNDMINGTKLLNVTKMTRGKRDGILRSEKYRKVVKIGSMHLKGVWIPFERALFIAKREKIVDLLYPLF
>5499_YARLI XP_505499 080..165
IIWDYHTGYVHLTGLWKAIGNSKADIVKLIDNSPDLEAVIRRVRGGYLKIQGTWVPYDIARALASRTCYFIRFALIPLFGQDFPGT
>5299_KLULA XP_455299 386..459
SVVRRADNDMINGTKLLNVTRMTRGRRDGILKAEKIRHVVKIGSMHLKGVWIPFERALVMAQREKIVDLLYALF
>0305_GIBZE XP_390305 226..298
VARREDNHMINGTKLLNVAGMTRGRRDGILKSEKVRHVVKIGPMHLKGVWIPYDRALDFANKEKITELLYPLF
>0837_NEUCR XP_960837 139..211
VARREDNAMINGTKLLNVAGMTRGRRDGILKSEKVRHVVKIGPMHLKGVWIPFERALDFANKEKITELLYPLF
>8552_MAGGR XP_368552 127..199
VARREDNHMINGTKLLNVAGMTRGRRDGILKSEKMRHVVKIGPMHLKGVWIPFERALDFANKEKITELLYPLF
>0447_DEBHA XP_460447 213..285
VSRREDTNYVNGTKLLNVAGMTRGKRDGILKTEKTKSVVKVGAMNLKGVWIPFERASEIARNEGIDGLLYPLF
>9978_GIBZE XP_389978 139..218
AVMWDYNIGLVRMTPFFKCRGYGKTIPAKMLGLNPGLKEITHSITGGSIAAQGYWMPYRCAKAICATFCHPIAGALIPIF
>1513_CANAL XP_711513 469..541
VSRREDTNYINGTKLLNVIGMTRGKRDGILKTEKIKNVVKVGSMNLKGVWIPFDRAYEIARNEGVDSLLYPLF
>6132_SCHPO NP_596132 088..165
LRRCPDSYFNISQILRLAGTSSSENAKELDDIIESGDYENVDSKHPQIDGVWVPYDRAISIAKRYGVYEILQPLISFN
>1244_ASPFU XP_751244 151..230
VMWDYNIGLVRTTHLFKCNDYSKMLNANPGLREICHSITGGALAAQGYWMPYEAAKAVAATFCWKIRHALTPLFGLDFPS
>0925_USTMA XP_760925 057..143
TMMIDVDTSFVRFTSITQALGKNKVNFGRLVKTCPALDPHITKLKGGYLSIQGTWLPFDLAKELSRRIAWEIRDHLVPLFGYDFPST
>2599_ASPTE XP_001212599 130..218
IMWDYNIGLVRTTPLFRSQNYSKTTPAKVLDANPGLREISHSITGGAIVAQDKPGYWIPFEAAKAVAATFCWRIRYALTPIFGLDFPSQ
>9773_DEBHA XP_459773 187..274
IIWDYETGFVHLTGIWKASINDEVNTHRNLKADIVKLLESTPKQYHQHIKRIRGGFLKIQGTWLPFDLCKMLAKRFCYHIRFQLIPIF
>0918_CANAL XP_710918 256..352
VIWDYETGWVHLTGIWKASLTIDGSNVSPSHLKADIVKLLESTPKEYQQYIKRIRGGFLKIQGTWLPYKLCKILARRFCYYLRYSLIPIFGTDFPDS
>9901_DEBHA XP_459901 067..158
ILRRVQDSYINISQLFSILLKIGHLSEAQLTNFLNNEILTNTQYLSSGGSNPQFNDLRNHEVRDLRGLWIPYDRAVSLALKFDIYELAKSLF
>7766_ASPNI XP_657766 089..163
LMRRSKDGYVSATGMFKIAFPWAKLEEERSEREYLKTRPETSEDEIAGNVWISPVLALELAAEYKMYDWVRALLD
>5459_GIBZE XP_385459 077..154
LMRRSYDGFVSATGMFKASFPYAEASDEDAERKYIKSLPTTSHEETAGNVWIPPEQALILAEEYKISPWIRALLDPTP
>2267_NEUCR XP_962267 085..162
LMRRSQDGYISATGMFKATFPYASQEEEEAERKYIKSIPTTSSEETAGNVWIPPEQALILAEEYQITPWIRALLDPSD
>3510_ASPFU XP_753510 089..163
LMRRSKDGYVSATGMFKIAFPWAKLEEEKAEREYLKTREGTSEDEIAGNIWVSPLLALELAKEYQMYDWVRALLD
>3762_MAGGR XP_363762 084..161
LMRRSSDGYVSATGMFKATFPYADAEDEEAERNYIKSLPATSKEETAGNVWISPDQALALAEEYSIATWIRALLDPTD
>3412_CANAL XP_723412 087..178
VLRRVQDSFVNVTQLFQILIKLEVLPTSQVDNYFDNEILSNLKYFGSSSNTPQYLDLRKHQNIYLQGIWIPYDKAVNLALKFDIYEITKKLF
>6166_SCHPO NP_596166 062..140
LMRMAKDSSISATSMFRSAFPKATQEEEDLEMRWIRDNLNPIEDKRVAGLWVPPADALALAKDYSMTPFINALLEASST
>XBP1_SACCE NP_012165 314..400
RDLICQSYKDFLINELGPDQIDLPNLNPANFTKRIRGGYIKIQGTWLPMEISRLLCLRFCFPIRYFLVPIFGPDFPKDCESWYLAHQ
>6355_ASPTE XP_001216355 084..167
TYFLMDGYVSATGMFKIAFPWAKLDEERSEREYLKSREETSEDEIAGNVWISPKLALELAGEYQMYNWVRALLDPTDIVQSPS
>9301_MAGGR XP_369301 092..188
EEYTVMWDYGCGLVRMTHFFKCRGYTKTVPGKVLNQNHGLKDITYSITGGSISAQESPNFGRMVIDRELVAHATREAESMYGRSMQAQAQQQGPLR
>5262_KLULA XP_455262 301..388
QQKWNKWFQRESFSTYIDLHWHKLNPTLSTLLGQSYDAKIPFERMVKRIRGGYIKIQGTWLPYPVSKELCSRFCYPLRYLLVPLFGPDFPEKCEYWY
>3869_EREGO NP_983869 277..365
YTDVHWNQVDPTWKQRLCRLYQQEKNLDFTPEFQDCYKRIRGGYIKIQGTWLPMEICKRLCIRFCFPIRYFLVPIFGEGFLQECHNWYF
>6482_CANGL XP_446482 300..390
SVNYLDFHWFDISEKVRSQIFEQFKQHLEKDRNVDCSTIPKAEEYIQRIRGGYIKIQGTWVPWYIAKLICIRFCFPIRYLLVPIFGEQFPV