ASPA Sequence Alignments
- 1 Sequence Searches
- 2 Multiple Sequence Alignments
- 3 Multiple Alignments
Prior to running BLAST, we concatenated all of the individual FASTA files of the nonredundant library into one big file, /data/nr/nr. Since we are working with proteins, BlastP was used. Command: blast -d /data/nr/nr -p blastp -i ../seq.fasta
We had to download and build this before being able to using it. Command: fasta36 -q ../seq.fasta /data/nr/nr
The preinstalled version of PSI Blast died on an error message; to fix this, we replaced /bin/sh with a symlink to /bin/bash.
blastpgp -i ../seq.fasta -d /data/nr/nr -j 3 -h 0.00001
blastpgp -i ../seq.fasta -d /data/nr/nr -j 5 -h 0.00001
blastpgp -i ../seq.fasta -d /data/nr/nr -j 3 -h 0.005
blastpgp -i ../seq.fasta -d /data/nr/nr -j 5 -h 0.005
Runtime for this was surprisingly long at approx. 23 minutes for 3 iterations and ca. 54 minutes for 5 iterations.
We used the downloadable HHSearch binary and the latest snapshot of the HMM database for PDB from ftp://toolkit.lmb.uni-muenchen.de/HHsearch/databases. Since this database diverges rather strongly from NR in its composition, we did not includee HHSearch results in any comparisons. The downloaded database (we used the HMM version) had to be concatenated into one single file before HHSearch would accept it as database input; this we did using a small Ruby script.
To obtain e-values, our input sequence had to be converted to a hhm file and calibrated against a calibration data set provided on the HHSearch website. This was done via the following commands:
hhmake -i seq.fasta
hhsearch -d cal.hhm -i seq.hhm -cal
We then searched the downloaded HMM database using the newly generated HHM profile for our input sequence:
Command: hhsearch -i ../seq.hhm -d ../hhsearch-db/pdb/db -o hhsearch-ali -p 0 -E 1000 -Z 100000000000 -B 10000000000
By specifying extreme values for p-value threshold, e-value cutoff and the minimum number of sequences to be shown in alignment and hit list, we ensured that low-identity matches were being reported too.
We visualize overlap of the results between different programs and settings using Venn diagrams; HHSearch is not included, as the difference in database composition precludes meaningful comparison. For a comparison between Blast, Fasta and PSI-Blast, we chose the PSI-Blast run with the least differences to the other PSI-Blast runs. In this particular case, the difference is fully negligible.
Multiple Sequence Alignments
Sequences selected from Sequence Searches
We selected the following sequences for the MSA task:
For building the multiple Alignments the following 20 sequences were chosen:
|99-90% Sequence Identity|
|197102934||93%||Aspartoacyclase (Pongo abelii)|
|296476730||92%||Aspartoacyclase (Bos taurus)|
|178056458||92%||Aspartoacyclase (Sus scrofa)|
|62510428||96%||Aspartoacyclase (Macaca fascicularis)|
|60-89% Sequence Identity|
|19354304||82%||Aspartoacyclase (Mus musculus)|
|13242314||81%||Aspartoacyclase (Rattus norvegicus)|
|166796542||67%||LOC733935 protein [Xenopus (Silurana) tropicalis]|
|227437329||65%||Aspartoacyclase (Hypomesus transpacificus)|
|158534041||62.5%||Aspartoacyclase (Danio rerio)|
|2jxm_B||60%||Cytochrome F (Prochlorothrix hollandica)|
|40-59% Sequence Identity|
|57526957||45%||Aspartoacyclase 2 (Rattus norvegicus)|
|89076336||40%||Aspartoacyclase (Photobacterium sp.)|
|18087825||41%||Aspartoacyclase 2 (Homo sapiens)|
|21309896||42%||Hepatitis C virus core-binding protein|
|308321821||42%||Aspartoacyclase 2A (Ictalurus furcatus)|
|20-39% Sequence Identity|
|88807290||34%||Aspartoacyclase (Synechococcus sp.)|
|158339037||37%||Aspartoacyclase (Acarychloris marina)|
|186683012||37%||Aspartoacyclase (Nostoc punctiforme)|
|308050175||37%||Aspartoacyclase (Ferrimonas balearica)|
|260771522||34%||Aspartoacyclase (Vibrio furnissii)|
Both Cytochrome F and Hepatitis C do not make much sense in this collection; however, we could not find any other more suitable sequences in our search results and therefore decided to include those to see what would happen; as it turns out, Cytochrome F affects the MSAs rather strongly, whereas Hepatitis C does not seem to have much impact and actually seems to have a surprisingly strong similarity to the aspartoacyclase sequences.
Command: clustalw selected.fasta
We had to install cobalt from the web; this being done, we ran it using the following command:
cobalt -i selected.fasta -norps T
Command: muscle -in selected.fasta -out msa_muscle.aln
T-Coffee (default parameters)
Command: t_coffee selected.fasta -outfile msa_tcoffee.aln
The corresponding mode for this flavor is expresso; it was already preinstalled on the VM used for the practical.
Command: t_coffee selected.fasta -outfile msa_tcoffee_3d.aln -mode expresso
Multiple Sequence Alignments
These are the MSAs that were created in this sub-task; the images were created using JalView.
Number of conserved columns
|Method||Number of conserved columns||Comment|
|Cobalt||7||Can be increased to 31 by ignoring the Cytochrome F sequence|
|ClustalW||3||Can be increased to 41 by ignoring Cytochrome F and Hypomesus transpacificus Aspartoacyclase|
|Muscle||2||Can be increased to 42 by ignoring Cytochrome F|
|T-Coffee||6||Can be increased to 89 by ignoring Cytochrome F|
|3D-Coffee||1||Can be increased to 45 by ignoring Cytochrome F|
The effect of Cytochrome F came as no surprise; what is surprising in this context is that we could not (at least without recalculating the alignments) find such effects with Hepatitis C.
Conservation of functionally important residues
The putative glycosylating residue is the aspartic acid at position 117. We checked two residues to the left and right for mutations. In three, 88807290, 158339037 and 186683012, sequences the functional residue was replaced with valin. Since all of them had sequence identities of 34-37% only, this is not especially surprising.
In 2JXM, the functional site was found to be quite completely destroyed. This is interesting since 2JXM was extracted from the Prochlorothrix hollandica algae, whereas some of the other proteins actually are of bacterial origin. We assume that 2JXM is not a true homolog and therefore slightly out of place in these alignments.
Number of Gaps
Gaps in Secondary Structure Elements