ExPSIBLAST - Revision history

Henni: /* Introduction */

2025-11-10T14:11:30Z

Introduction

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	<!-- * [https://services.healthtech.dtu.dk/service.php?Blast2logo Blast2logo] is a tool for visualization of protein sequence profiles and identification of conserved residues.		<!-- * [https://services.healthtech.dtu.dk/service.php?Blast2logo Blast2logo] is a tool for visualization of protein sequence profiles and identification of conserved residues.
	-->		-->

			===Technical issues===
			If you are denied access to the BLAST server, it may help to make a hotspot on your phone and use that to connect to the internet instead of the DTU Wi-Fi.

			In case you cannot make the BLAST server run at all, we have made some "backup output" links with copies of the relevant BLAST output page. '''Note:''' The backup outputs ''cannot'' show <u>Graphic Summary</u> or <u>Alignments</u>!

	==When BLAST fails==		==When BLAST fails==

Henni: /* Finding a remote homolog (on your own) */

2025-11-10T14:03:59Z

Finding a remote homolog (on your own)

← Older revision		Revision as of 16:03, 10 November 2025
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	PSI-Blast is not only useful for finding a remote homolog in a specific database such as PDB — now it is time to search the broader database "Reference proteins" (<u>refseq_protein</u>). ('''Note:''' we would have liked to do this exercise in the broadest database <u>nr</u>, but that search runs into technical problems). PSI-BLAST can be used in the same way for finding a remote homolog in a specific organism or taxonomic group. Your task in this round is to find out whether the protein with the UniProt ID '''GPAA1_HUMAN''' has a homolog in the genus ''Trypanosoma'' (unicellular parasites which cause diseases like sleeping sickness or Chaga's disease).		PSI-Blast is not only useful for finding a remote homolog in a specific database such as PDB — now it is time to search the broader database "Reference proteins" (<u>refseq_protein</u>). ('''Note:''' we would have liked to do this exercise in the broadest database <u>nr</u>, but that search runs into technical problems). PSI-BLAST can be used in the same way for finding a remote homolog in a specific organism or taxonomic group. Your task in this round is to find out whether the protein with the UniProt ID '''GPAA1_HUMAN''' has a homolog in the genus ''Trypanosoma'' (unicellular parasites which cause diseases like sleeping sickness or Chaga's disease).
	* First, try a standard BlastP (where you set <u>Organism</u> to ''Trypanosoma'', <u>Database</u> to <u>refseq_protein</u> ('''not''' refseq_select), switch the <u>Low complexity regions</u> filter off, and set the E-value threshold to 10).		* First, try a standard BlastP (where you set <u>Organism</u> to ''Trypanosoma'', <u>Database</u> to <u>refseq_protein</u> ('''not''' refseq_select), switch the <u>Low complexity regions</u> filter off, and set the E-value threshold to 10).
	* '''QUESTION 13''': Do you find any significant (E<0.005) hits? What is the E-value of the best hit?		* '''QUESTION 13''': Do you find any significant (E<0.005) hits? What is the E-value of the best hit? ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q13.html backup output])
	* Then, try PSI-BLAST. '''Hint:''' You need to search in all organisms (still using refseq_protein) to build a PSSM, then save your PSSM and use that to search in ''Trypanosoma''.		* Then, try PSI-BLAST. '''Hint:''' You need to search in all organisms (still using refseq_protein) to build a PSSM, then save your PSSM and use that to search in ''Trypanosoma''.
	* '''QUESTION 14''': How many significant (E<0.005) hits do you find now? What is the E-value of the best hit?		* '''QUESTION 14''': How many significant (E<0.005) hits do you find now? What is the E-value of the best hit? ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q14A.html backup output 1]) ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q14-PSSM_Scoremat.asn backup PSSM]) ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q14B.html backup output 2])


	<!--		<!--

Henni: /* Trying another approach */

2025-11-10T13:58:36Z

Trying another approach

Henni: /* When BLAST fails */

2025-11-10T13:48:21Z

When BLAST fails

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	* '''QUESTION 1''': How many significant hits does BLAST find (E-value < 0.005)?		* '''QUESTION 1''': How many significant hits does BLAST find (E-value < 0.005)? ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q1.html backup output])

	==Trying another approach==		==Trying another approach==

Henni: /* One more round */

2025-11-10T08:52:36Z

One more round

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	===One more round===		===One more round===
	Let's try one more iteration of PSI-BLAST:		Let's try one more iteration of PSI-BLAST:
	* Go back to your first BLAST window (the one with the results from the <u>nr</u> database limited to Archaea) and press the <u>Run</u> button at <u>Run PSI-Blast iteration 3</u>.		* Go back to your first BLAST window (the one with the results from the <u>refseq_protein</u> database limited to Archaea) and press the <u>Run</u> button at <u>Run PSI-Blast iteration 3</u>.
	* Save the resulting PSSM file (make sure you give it a different name!).		* Save the resulting PSSM file (make sure you give it a different name!).
	* Launch a new PSI-BLAST search against <u>pdb</u> in all organisms using this PSSM (you may have to click on <u>Clear</u> to erase your first PSSM file from the server).		* Launch a new PSI-BLAST search against <u>pdb</u> in all organisms using this PSSM (you may have to click on <u>Clear</u> to erase your first PSSM file from the server).

Henni: /* Saving and reusing the PSSM */

2025-11-09T14:19:05Z

Saving and reusing the PSSM

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	Then, open ''a new BLAST window'' (this is important—you need your first BLAST window again later) where you again select PSI-BLAST as the algorithm. Select <u>pdb</u> as the database. Do ''not'' limit your search to Archaea this time. Click on <u>Algorithm parameters</u> to show the extended settings. Click the button next to <u>Upload PSSM</u> and select the file you just saved. '''Note:''' You don't have to paste the query sequence again, it is stored in the PSSM!		Then, open ''a new BLAST window'' (this is important—you need your first BLAST window again later) where you again select PSI-BLAST as the algorithm. Select <u>pdb</u> as the database. Do ''not'' limit your search to Archaea this time. Click on <u>Algorithm parameters</u> to show the extended settings. Click the button next to <u>Upload PSSM</u> and select the file you just saved. '''Note:''' You don't have to paste the query sequence again, it is stored in the PSSM!

	* '''QUESTION 8''': Do you find any significant PDB hits now? If yes, how many?		* '''QUESTION 8''': Do you find any significant PDB hits (E-value < 0.005) now? If yes, how many?
	* '''QUESTION 9''': What are the PDB identifiers and the E-values for the two best PDB hits?		* '''QUESTION 9''': What are the PDB identifiers and the E-values for the two best PDB hits?
	* '''QUESTION 10''': What are the values for Query coverage, sequence identity, and sequence similarity (Positives) for the two best PDB hits? ('''Tip:''' click on the description to get to the actual alignment between the query sequence and the PDB hit)?		* '''QUESTION 10''': What are the values for Query coverage, sequence identity, and sequence similarity (Positives) for the two best PDB hits? ('''Tip:''' click on the description to get to the actual alignment between the query sequence and the PDB hit)?

Henni: /* Constructing the PSSM */

2025-11-09T13:27:15Z

Constructing the PSSM

← Older revision		Revision as of 15:27, 9 November 2025
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	:'''Note:''' If you see the error message “<u>Entrez Query: txid2157 [ORGN] is not supported</u>”, then click <u>Recent Results</u> in the upper right part of the BLAST window, select your most recent search, and try again.		:'''Note:''' If you see the error message “<u>Entrez Query: txid2157 [ORGN] is not supported</u>”, then click <u>Recent Results</u> in the upper right part of the BLAST window, select your most recent search, and try again.
	</div>		</div>
	Now run a second BLAST iteration in order to construct a PSSM (Position-Specific Scoring Matrix). Press the <u>Run</u> button at <u>Run PSI-Blast iteration 2</u> (you can find it at ~~both~~ the ~~bottom and~~ top of the results table).		Now run a second BLAST iteration in order to construct a PSSM (Position-Specific Scoring Matrix). Press the <u>Run</u> button at <u>Run PSI-Blast iteration 2</u> (you can find it both at the top of the results table and after the list of significant hits).

	* '''QUESTION 4''': How many significant hits does BLAST find (E-value < 0.005)?		* '''QUESTION 4''': How many significant hits does BLAST find (E-value < 0.005)?

Henni: /* Trying another approach */

2025-11-08T16:18:49Z

Trying another approach

← Older revision		Revision as of 18:18, 8 November 2025
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	* '''QUESTION 2''': How many significant hits does BLAST find (E-value < 0.005)? ('''Tip:''' you can see the number by selecting all significant hits (clicking <u>All</u> under <u>Sequences with E-value BETTER than threshold</u>) and then looking at the number of selected hits)		* '''QUESTION 2''': How many significant hits does BLAST find (E-value < 0.005)? ('''Tip:''' you can see the number by selecting all significant hits (clicking <u>All</u> under <u>Sequences with E-value BETTER than threshold</u>) and then looking at the number of selected hits)
	* '''QUESTION 3''': How large a fraction (Query coverage) of the query sequence do the significant hits typically match (excluding the 100% identity match)?		* '''QUESTION 3''': How large a fraction (Query coverage) of the query sequence do the significant hits typically match (excluding the 100% identity match)?
	* '''QUESTION 4''': (deleted) ~~<!--~~ Do you find any PDB hits among the significant hits? ('''Tip:''' look for a PDB identifier in the <u>Accession</u> column — a PDB identifier is a 4 character code, where the first character is a number, followed by a single letter chain name, such as "1XYZ_A") -->		<!-- * '''QUESTION 4''': (deleted because it only makes sense when using nr) Do you find any PDB hits among the significant hits? ('''Tip:''' look for a PDB identifier in the <u>Accession</u> column — a PDB identifier is a 4 character code, where the first character is a number, followed by a single letter chain name, such as "1XYZ_A") -->

	===Constructing the PSSM===		===Constructing the PSSM===
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	Now run a second BLAST iteration in order to construct a PSSM (Position-Specific Scoring Matrix). Press the <u>Run</u> button at <u>Run PSI-Blast iteration 2</u> (you can find it at both the bottom and top of the results table).		Now run a second BLAST iteration in order to construct a PSSM (Position-Specific Scoring Matrix). Press the <u>Run</u> button at <u>Run PSI-Blast iteration 2</u> (you can find it at both the bottom and top of the results table).

	* '''QUESTION 5''': How many significant hits does BLAST find (E-value < 0.005)?		* '''QUESTION 4''': How many significant hits does BLAST find (E-value < 0.005)?
	* '''QUESTION 6''': How large a fraction of the query sequence do the 20 most significant hits match (do not include the ~~first hit since this is identical to the query~~)?		* '''QUESTION 5''': What is the E-value of the ''least'' significant hit shown on the results page?
			* '''QUESTION 6''': How large a fraction of the query sequence do the 20 most significant hits match (do not include the 100% identity match)?
	* '''QUESTION 7''': Why does BLAST come up with more significant hits in the second iteration? Make sure you answer this question and understand what is going on!		* '''QUESTION 7''': Why does BLAST come up with more significant hits in the second iteration? Make sure you answer this question and understand what is going on!

Henni: /* Trying another approach */

2025-11-08T15:53:44Z

Trying another approach

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	==Trying another approach==		==Trying another approach==

	Now go back to the search web-site of [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome BLASTP]. ~~Paste~~ in the query sequence again. This time, set the database to <u>~~Non-redundant protein sequences~~ (nr)</u> and select <u>PSI-BLAST (Position-Specific Iterated BLAST)</u> as the algorithm (Figure 2).		Now, <!-- go back to the search web-site of [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome BLASTP].--> click on <u>Edit Search</u> on the results page (then you don't have to paste in the query sequence again). This time, set the database to <u>Reference proteins (refseq_protein)</u> and select <u>PSI-BLAST (Position-Specific Iterated BLAST)</u> as the algorithm (Figure 2).

	'''IMPORTANT:''' To ease the load on the NCBI server, limit the search to Archaea (TaxID 2157) when searching Query1 in nr.		'''IMPORTANT:''' To ease the load on the NCBI server, limit the search to Archaea (TaxID 2157) when searching Query1 in refseq_protein. We know that the mysterious "Query1" sequence is from an archaeon.


	[[File:psiblastp_nr.png\|250px\|center\|frame\|Figure 2. Partial screenshot of the PSI-BLAST interface. The red arrow shows the settings change to PSI-BLAST.]]		[[File:psiblastp_nr.png\|250px\|center\|frame\|Figure 2. Partial screenshot of the PSI-BLAST interface. The red arrow shows the settings change to PSI-BLAST.]]

	* '''QUESTION 2''': How many significant hits does BLAST find (E-value < 0.005)? ('''Tip:''' you can see the number by selecting all significant hits (clicking <u>All</u> under <u>Sequences ~~producing significant alignments~~ with E-value BETTER than threshold</u>) and then looking at the number of selected hits)		* '''QUESTION 2''': How many significant hits does BLAST find (E-value < 0.005)? ('''Tip:''' you can see the number by selecting all significant hits (clicking <u>All</u> under <u>Sequences with E-value BETTER than threshold</u>) and then looking at the number of selected hits)
	* '''QUESTION 3''': How large a fraction (Query coverage) of the query sequence do the significant hits match (excluding the ~~identical~~ match)?		* '''QUESTION 3''': How large a fraction (Query coverage) of the query sequence do the significant hits typically match (excluding the 100% identity match)?
	* '''QUESTION 4''': Do you find any PDB hits among the significant hits? ('''Tip:''' look for a PDB identifier in the <u>Accession</u> column — a PDB identifier is a 4 character code, where the first character is a number, followed by a single letter chain name, such as "1XYZ_A")		* '''QUESTION 4''': (deleted) <!-- Do you find any PDB hits among the significant hits? ('''Tip:''' look for a PDB identifier in the <u>Accession</u> column — a PDB identifier is a 4 character code, where the first character is a number, followed by a single letter chain name, such as "1XYZ_A") -->

	===Constructing the PSSM===		===Constructing the PSSM===

Henni: /* When BLAST fails */

2025-11-08T15:28:20Z

When BLAST fails

← Older revision		Revision as of 17:28, 8 November 2025
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	==When BLAST fails==		==When BLAST fails==

	Say you have a protein sequence [https://teaching.healthtech.dtu.dk/material/22111/files/Query1.txt Query] (also pasted below), and you want to ~~make predictions about its structural~~ homologue. As seen earlier in the course, you will most often use BLAST to do this. However what happens when BLAST fails?		Say you have a protein sequence [https://teaching.healthtech.dtu.dk/material/22111/files/Query1.txt Query] (also pasted below), and you want to find a homologue with experimentally known structure. As seen earlier in the course, you will most often use BLAST to do this. However what happens when BLAST fails?

	>QUERY1		>QUERY1
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	Go to the [http://www.ncbi.nlm.nih.gov/BLAST BLAST] web-site at NCBI. Select <u>blastp</u> as the algorithm. Paste in the query sequence. Change the database from nr to <u> Protein Data Bank (pdb)</u>, and press <u>BLAST</u> (Figure 1).		Go to the [http://www.ncbi.nlm.nih.gov/BLAST BLAST] web-site at NCBI. Select <u>blastp</u> as the algorithm. Paste in the query sequence. Change the database from <u>ClusteredNR</u> to <u>Protein Data Bank (pdb)</u>, and press <u>BLAST</u> (Figure 1).

	[[File:blastp_pdb.png\|center\|frame\|Figure 1. Partial screenshot of the Blast interface. The red arrow shows the settings change to the database to pdb]]		[[File:blastp_pdb.png\|center\|frame\|Figure 1. Partial screenshot of the Blast interface. The red arrow shows the settings change to the database to pdb]]

← Older revision		Revision as of 15:58, 10 November 2025
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	[[File:psiblastp_nr.png\|250px\|center\|frame\|Figure 2. Partial screenshot of the PSI-BLAST interface. The red arrow shows the settings change to PSI-BLAST.]]		[[File:psiblastp_nr.png\|250px\|center\|frame\|Figure 2. Partial screenshot of the PSI-BLAST interface. The red arrow shows the settings change to PSI-BLAST.]]

	* '''QUESTION 2''': How many significant hits does BLAST find (E-value < 0.005)? ('''Tip:''' you can see the number by selecting all significant hits (clicking <u>All</u> under <u>Sequences with E-value BETTER than threshold</u>) and then looking at the number of selected hits)		* '''QUESTION 2''': How many significant hits does BLAST find (E-value < 0.005)? ('''Tip:''' you can see the number by selecting all significant hits (clicking <u>All</u> under <u>Sequences with E-value BETTER than threshold</u>) and then looking at the number of selected hits) ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q2.html backup output])
	* '''QUESTION 3''': How large a fraction (Query coverage) of the query sequence do the significant hits typically match (excluding the 100% identity match)?		* '''QUESTION 3''': How large a fraction (Query coverage) of the query sequence do the significant hits typically match (excluding the 100% identity match)?
	<!-- * '''QUESTION 4''': (deleted because it only makes sense when using nr) Do you find any PDB hits among the significant hits? ('''Tip:''' look for a PDB identifier in the <u>Accession</u> column — a PDB identifier is a 4 character code, where the first character is a number, followed by a single letter chain name, such as "1XYZ_A") -->		<!-- * '''QUESTION 4''': (deleted because it only makes sense when using nr) Do you find any PDB hits among the significant hits? ('''Tip:''' look for a PDB identifier in the <u>Accession</u> column — a PDB identifier is a 4 character code, where the first character is a number, followed by a single letter chain name, such as "1XYZ_A") -->
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	Now run a second BLAST iteration in order to construct a PSSM (Position-Specific Scoring Matrix). Press the <u>Run</u> button at <u>Run PSI-Blast iteration 2</u> (you can find it both at the top of the results table and after the list of significant hits).		Now run a second BLAST iteration in order to construct a PSSM (Position-Specific Scoring Matrix). Press the <u>Run</u> button at <u>Run PSI-Blast iteration 2</u> (you can find it both at the top of the results table and after the list of significant hits).

	* '''QUESTION 4''': How many significant hits does BLAST find (E-value < 0.005)?		* '''QUESTION 4''': How many significant hits does BLAST find (E-value < 0.005)? ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q4.html backup output])
	* '''QUESTION 5''': What is the E-value of the ''least'' significant hit shown on the results page?		* '''QUESTION 5''': What is the E-value of the ''least'' significant hit shown on the results page?
	* '''QUESTION 6''': How large a fraction of the query sequence do the 20 most significant hits match (do not include the 100% identity match)?		* '''QUESTION 6''': How large a fraction of the query sequence do the 20 most significant hits match (do not include the 100% identity match)?
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	===Saving and reusing the PSSM===		===Saving and reusing the PSSM===
	This time, we will not ask you to look for PDB identifiers manually among the significant hits. Instead, you should save the PSSM that PSI-BLAST has created and use it for searching PDB directly.		<!-- This time, we will not ask you to look for PDB identifiers manually among the significant hits. Instead, you should save the PSSM that PSI-BLAST has created and use it for searching PDB directly.-->
			Now, you are going to save the PSSM that PSI-BLAST has created and use it for searching PDB.

	Go to the top of the PSI-BLAST output page and click <u>Download All</u>, then click <u>PSSM</u>. Save the file to a place on your computer where you can find it again! You can take a look at this file using Geany, but it is really not meant to be human-readable.		Go to the top of the PSI-BLAST output page and click <u>Download All</u>, then click <u>PSSM</u>. Save the file to a place on your computer where you can find it again! You can take a look at this file using Geany, but it is really not meant to be human-readable. ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q4-PSSM_Scoremat.asn backup PSSM])

	Then, open ''a new BLAST window'' (this is important—you need your first BLAST window again later) where you again select PSI-BLAST as the algorithm. Select <u>pdb</u> as the database. Do ''not'' limit your search to Archaea this time. Click on <u>Algorithm parameters</u> to show the extended settings. Click the button next to <u>Upload PSSM</u> and select the file you just saved. '''Note:''' You don't have to paste the query sequence again, it is stored in the PSSM!		Then, open ''a new BLAST window'' (this is important—you need your first BLAST window again later) where you again select PSI-BLAST as the algorithm. Select <u>pdb</u> as the database. Do ''not'' limit your search to Archaea this time. Click on <u>Algorithm parameters</u> to show the extended settings. Click the button next to <u>Upload PSSM</u> and select the file you just saved. '''Note:''' You don't have to paste the query sequence again, it is stored in the PSSM!

	* '''QUESTION 8''': Do you find any significant PDB hits (E-value < 0.005) now? If yes, how many?		* '''QUESTION 8''': Do you find any significant PDB hits (E-value < 0.005) now? If yes, how many? ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q8.html backup output])
	* '''QUESTION 9''': What are the PDB identifiers and the E-values for the two best PDB hits?		* '''QUESTION 9''': What are the PDB identifiers and the E-values for the two best PDB hits?
	* '''QUESTION 10''': What are the values for Query coverage, sequence identity, and sequence similarity (Positives) for the two best PDB hits? ('''Tip:''' click on the description to get to the actual alignment between the query sequence and the PDB hit)?		* '''QUESTION 10''': What are the values for Query coverage, sequence identity, and sequence similarity (Positives) for the two best PDB hits? ('''Tip:''' click on the description to get to the actual alignment between the query sequence and the PDB hit)?
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	===One more round===		===One more round===
	Let's try one more iteration of PSI-BLAST:		Let's try one more iteration of PSI-BLAST:
	* Go back to your first BLAST window (the one with the results from the <u>refseq_protein</u> database limited to Archaea) and press the <u>Run</u> button at <u>Run PSI-Blast iteration 3</u>.		* Go back to your first BLAST window (the one with the results from the <u>refseq_protein</u> database limited to Archaea) and press the <u>Run</u> button at <u>Run PSI-Blast iteration 3</u>. ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q12A.html backup output])
	* Save the resulting PSSM file (make sure you give it a different name!).		* Save the resulting PSSM file (make sure you give it a different name!). ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q12-PSSM_Scoremat.asn backup PSSM])
	* Launch a new PSI-BLAST search against <u>pdb</u> in all organisms using this PSSM (you may have to click on <u>Clear</u> to erase your first PSSM file from the server).		* Launch a new PSI-BLAST search against <u>pdb</u> in all organisms using this PSSM (you may have to click on <u>Clear</u> to erase your first PSSM file from the server).
	* '''QUESTION 12''': Answer questions 8-10 again for the new search.		* '''QUESTION 12''': Answer questions 8-10 again for the new search. ([https://teaching.healthtech.dtu.dk/material/22111/PSI-BLAST/Q12B.html backup output])

	==Finding a remote homolog (on your own)==		==Finding a remote homolog (on your own)==