Tutorial: Blast a sequence against TCDB
- This tutorial shows how to use our TC-BLAST page in order scan all proteins in TCDB for homologs of any query sequence. Internally, TC-Blast runs the program BlastP from NCBI. All search parameters in this page are a subset of those used in the official NCBI Blast page.
Anatomy of the tool:
In this example, we will blast the sequence of protein P68183 (TCID: 3.A.1.1.1) against TCDB. The Blast parameters that we will use for this search are shown in Figure 1. For your convenience, here is the sequence that you will copy and then paste into the input form (Figure 1I):
>P68183|MALG_ECOLI Maltose transport system permease protein malG - Escherichia coli MAMVQPKSQKARLFITHLLLLLFIAAIMFPLLMVVAISLRQGNFATGSLIPEQISWDHWK LALGFSVEQADGRITPPPFPVLLWLWNSVKVAGISAIGIVALSTTCAYAFARMRFPGKAT LLKGMLIFQMFPAVLSLVALYALFDRLGEYIPFIGLNTHGGVIFAYLGGIALHVWTIKGY FETIDSSLEEAAALDGATPWQAFRLVLLPLSVPILAVVFILSFIAAITEVPVASLLLRDV NSYTLAVGMQQYLNPQNYLWGDFAAAAVMSALPITIVFLLAQRWLVNGLTAGGVKGE-value cutoff to use. C. Set the maximal number of match descriptions to include in the results. D. Set maximal number of alignments to include in the results. E. Set the Amino acid substitution matrix to use. F. Set the gap opening penalty. G. Set the gap extension penalty. H. Filter out low complexity regions that might cause misleading results. I. Input box for query sequence. J. Run BlastP against TCDB. K. Reset all parameters to defaults.
Paste your sequence on the input box (Figure 1I) as shown in Figure 2, and select the search parameters of your preference. For example, you can select a more stringent E-value (Figure 1B) if you are searching for close homologs of your query sequence, or activate the low complexity filter (Figure 1H). However, use this option carefully if you are interested in transmembranal regions, as these regions may have small repetitive patches of hydrophobic residues that would be ignored by the low complexity filter (which may not be your intention). Once you are ready, submit your query by clicking on the button "Click here to BLAST" (Figure 1J).Figure 2. Blast your query sequence against all proteins in TCDB.
Step 2: Review and analyze the results.
The BLAST output consists, first, of a graphical display that shows the regions in the query sequence predicted as TMSs (Figure 3A) using the program HMMTOP, and the regions covered by the top alignments between your query protein and the matched TCDB proteins (Figure 3B). This will give you an idea of how good the matches are in terms of their coverage relative to your query protein (Figure 3). Second, a list of the top TCDB matches, sorted by E-value, showing links to the specific system entries matching your query (Figure 4A), their number of TMSs (Figure 4B), their TCID linked to their family entry (Figure 4C), a brief description (Figure 4D), the bit score linked to the corresponding alignment in the same page (Figure 4E), and the alignment E-value (Figure 4F).
Figure 3. BLAST graphical output. A. Location of predicted transmembrane segments in the query protein. B. Regions of the query protein covered by the alignments with matched TCDB proteins.
Figure 4. List of BLAST matches. A. Protein accession. B. Number of TMSs in matched tcdb proteins. C. TCID. D. Description. E. Bit score. F. E-value.
Click on the accession(s) (Figure 4A) of the homolog proteins you are interested in to go to the TC system entry. If the TCDB protein you select is a member of a multi-component system, the linked page will show all the proteins in that system (Figure 5). In this page, just click again on the accession you were originally interested in (Figure 4A and Figure 5A). In this example, we select the protein with accession P68183.
Figure 5. Multi-component system 3.A.1.1.1 (ABC) Superfamily. A. Particular component of interest.
For details about the functionality of the header of the page shown in Figure 5, follow our tutorials: The "Search TCDB" box and The TCDB homepage. For a detailed description of the page you will be directed to after clicking on the component of interest (Figure 5A), please, follow our tutorial: The system component entry page.