Exercise PSI-BLAST ans - Revision history

Carol at 12:26, 6 November 2025

2025-11-06T12:26:48Z

← Older revision		Revision as of 14:26, 6 November 2025
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	The first two hit proteins on PDB (5N07_A and 5N08_A) are called HTH-type transcriptional repressor NsrR. These proteins bind DNA with the Helix-turn-Helix motif (HTH) to repress the transcription of genes, but that part of the protein is outside of our alignment so it is not a function that would be present in our orphan protein.		The first two hit proteins on PDB (5N07_A and 5N08_A) are called HTH-type transcriptional repressor NsrR. These proteins bind DNA with the Helix-turn-Helix motif (HTH) to repress the transcription of genes, but that part of the protein is outside of our alignment so it is not a function that would be present in our orphan protein.
	Additionally there is some additional information on Uniprot about this protein (Q9L132) Binds DNA; this binding is disrupted by nitrosylation upon exposure to nitric oxide (NO) and also by EDTA and iron chelators. The 2Fe-2S cluster is stable in the presence of O2. This regulatory function is dependent on three Cysteines (C) that bind iron. Interestingly we also found the three cysteins in our protein of interest so might have some regulatory function.. but at this point we will probably need to do some experimental assays to test this hypothesis.		Additionally there is some additional information on Uniprot about this protein (Q9L132) Binds DNA; this binding is disrupted by nitrosylation upon exposure to nitric oxide (NO) and also by EDTA and iron chelators. The 2Fe-2S cluster is stable in the presence of O2. This regulatory function is dependent on three Cysteines (C) that bind iron. Interestingly we also found the three cysteins in our protein of interest so might have some regulatory function.. but at this point we will probably need to do some experimental assays to test this hypothesis.

			QUESTION 13: However, can you see any potential risks on doing so? Can we believe in the results?
			The Orphan protein used for the example is a real case scenario protein, unfortunately we do not know the function. There are still many genes that we do not know what they do, and some are involved in diseases so it is important to find ways to find a potential function for them.
			When we use PSI-BLAST we select some sequences to build a position-specific scoring matrix (PSSM). The advantages of using a matrix instead of a single sequence for remote homologous sequences has the advantage of learning a wider range of preferences for each position, and that is the reason why we find more hits and with lower (more significant) E-values.
			However, a cautious note, you should be sure that the sequences that you include in your PSSM will not polute the initial signal.. so preferentially they will come from a few or lower significant hits.

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

Carol at 12:11, 6 November 2025

2025-11-06T12:11:02Z

← Older revision		Revision as of 14:11, 6 November 2025
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	* '''QUESTION 12''': What is the function of these proteins?		* '''QUESTION 12''': What is the function of these proteins?
	The first two hit proteins on PDB (5N07_A and 5N08_A) are called HTH-type transcriptional repressor NsrR. These proteins bind DNA with the Helix-turn-Helix motif (HTH) to repress the transcription of genes, but that part of the protein is outside of our alignment so it is not a function that would be present in our orphan protein.		The first two hit proteins on PDB (5N07_A and 5N08_A) are called HTH-type transcriptional repressor NsrR. These proteins bind DNA with the Helix-turn-Helix motif (HTH) to repress the transcription of genes, but that part of the protein is outside of our alignment so it is not a function that would be present in our orphan protein.
	Additionally there is some additional information on Uniprot about this protein (Q9L132) Binds DNA; this binding is disrupted by nitrosylation upon exposure to nitric oxide (NO) and also by EDTA and iron chelators. The 2Fe-2S cluster is stable in the presence of O2. This regulatory function is dependent on three Cysteines (C) that bind iron. Interestingly we also found the three cysteins in our protein of interest so might have some regulatory function.. but at this point ~~seems a bit of speculation.. and~~ we will probably need to do some experimental assays to test ~~its function~~.		Additionally there is some additional information on Uniprot about this protein (Q9L132) Binds DNA; this binding is disrupted by nitrosylation upon exposure to nitric oxide (NO) and also by EDTA and iron chelators. The 2Fe-2S cluster is stable in the presence of O2. This regulatory function is dependent on three Cysteines (C) that bind iron. Interestingly we also found the three cysteins in our protein of interest so might have some regulatory function.. but at this point we will probably need to do some experimental assays to test this hypothesis.

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

Carol at 12:00, 6 November 2025

2025-11-06T12:00:42Z

← Older revision		Revision as of 14:00, 6 November 2025
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	* '''QUESTION 11''': Do you find any significant PDB hits now? Look at the Graphic Summary and query coverage. Is this what you expected, Why?		* '''QUESTION 11''': Do you find any significant PDB hits now? Look at the Graphic Summary and query coverage. Is this what you expected, Why?

			[[File:PSSM-2_on_PDB.png\|800px\|center]]

			[[File:graphicSummary_PSSM_onPDB.png\|800px\|center]]

	Answer: Both searching wiht PSSM-2 and PSSM-3 we can find the same matches on PDB. The coverage coincides with the second half (C-terminal) of the orphan protein. This result is expected since in our PSSM constructions we had more proteins from this region of the protein.. so we can see it as if we had amplified the signal from this region.		Answer: Both searching wiht PSSM-2 and PSSM-3 we can find the same matches on PDB. The coverage coincides with the second half (C-terminal) of the orphan protein. This result is expected since in our PSSM constructions we had more proteins from this region of the protein.. so we can see it as if we had amplified the signal from this region.

Carol at 11:59, 6 November 2025

2025-11-06T11:59:36Z

← Older revision		Revision as of 13:59, 6 November 2025
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	[[File:graphicSummary_PB3.png\|800px\|center]]		[[File:graphicSummary_PB3.png\|800px\|center]]

	Answer: The e-values are lower this time but the ~~query~~ cover seems to be skewed to only one part of the previous matches.		Answer: The e-values are lower this time and the query cover has increased to around 74% but the cover seems to be skewed to only one part of the previous matches. These suggests that only from the two types of matches one has dominated in the construction of the PSSM-3.

	* '''QUESTION 9''': Are there any homologous sequences found in search 2 that have an annotated function?		* '''QUESTION 9''': Are there any homologous sequences found in search 2 that have an annotated function?
	In the previous search (PSI-BLAST run 2) the functions were mostly deaminase domain-containing protein and Rrf2 family transcriptional regulator and some hypethical proteins with unknown function.		Answer: In the previous search (PSI-BLAST run 2) the functions were mostly deaminase domain-containing protein and Rrf2 family transcriptional regulator and some hypethical proteins with unknown function.

	* '''QUESTION 10''': Are there any homologous sequences found in search 3 that have an annotated function? Is there anything in common with search 2?		* '''QUESTION 10''': Are there any homologous sequences found in search 3 that have an annotated function? Is there anything in common with search 2?
	In the new search (PSI-BLAST run 3) the functions were mostly Rrf2 family transcriptional regulator		Answer: In the new search (PSI-BLAST run 3) the functions were mostly Rrf2 family transcriptional regulator, this also agrees with what we observed on the graphic summary showing the coverage it seems only the matches on one of the regions (C-terminal part of the protein) has dominated the PSSM-3.


	* '''QUESTION 11''': Do you find any significant PDB hits now? Look at the Graphic Summary and query coverage. Is this what you expected, Why?		* '''QUESTION 11''': Do you find any significant PDB hits now? Look at the Graphic Summary and query coverage. Is this what you expected, Why?
	* '''QUESTION 12''': What is the function of these proteins?

			Answer: Both searching wiht PSSM-2 and PSSM-3 we can find the same matches on PDB. The coverage coincides with the second half (C-terminal) of the orphan protein. This result is expected since in our PSSM constructions we had more proteins from this region of the protein.. so we can see it as if we had amplified the signal from this region.
			We could have selected only the other proteins for the second iteration of PSI-BLAST to skew the results to the other (N-terminal) portion of the protein. It also makes sense that the PDB structures are coming from Bacteria.

			* '''QUESTION 12''': What is the function of these proteins?
			The first two hit proteins on PDB (5N07_A and 5N08_A) are called HTH-type transcriptional repressor NsrR. These proteins bind DNA with the Helix-turn-Helix motif (HTH) to repress the transcription of genes, but that part of the protein is outside of our alignment so it is not a function that would be present in our orphan protein.
			Additionally there is some additional information on Uniprot about this protein (Q9L132) Binds DNA; this binding is disrupted by nitrosylation upon exposure to nitric oxide (NO) and also by EDTA and iron chelators. The 2Fe-2S cluster is stable in the presence of O2. This regulatory function is dependent on three Cysteines (C) that bind iron. Interestingly we also found the three cysteins in our protein of interest so might have some regulatory function.. but at this point seems a bit of speculation.. and we will probably need to do some experimental assays to test its function.

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

Carol at 11:12, 6 November 2025

2025-11-06T11:12:55Z

← Older revision		Revision as of 13:12, 6 November 2025
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	~~NEW answers are being updated!~~

	* '''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)?
	Answer: No sequences with E-value below 0.005.		Answer: No sequences with E-value below 0.005.
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	* '''QUESTION 5''': After iteration 2, How many significant hits (E-value < 0.005) are now found? What happened with E-value of the hits found before?		* '''QUESTION 5''': After iteration 2, How many significant hits (E-value < 0.005) are now found? What happened with E-value of the hits found before?

			[[File:results_PSI-BLAST_iteration2.png\|800px\|center]]

	Answer: 500 (actually, much more than 500 hits, but BLAST by default only shows 500 — note that the last hit has an E-value much much smaller than 0.005). The E-values of the previous hits are much lower and look significant this time. This is because those sequences were integrated on the PSSM and therefore on the search.		Answer: 500 (actually, much more than 500 hits, but BLAST by default only shows 500 — note that the last hit has an E-value much much smaller than 0.005). The E-values of the previous hits are much lower and look significant this time. This is because those sequences were integrated on the PSSM and therefore on the search.

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	* '''QUESTION 8''': Can you see any changes on the results now? Look at the E-values, and the query cover on the Graphic Summary tab.		* '''QUESTION 8''': Can you see any changes on the results now? Look at the E-values, and the query cover on the Graphic Summary tab.

			[[File:results_PSI-BLAST_iteration3.png\|800px\|center]]

			[[File:graphicSummary_PB3.png\|800px\|center]]

	Answer: The e-values are lower this time but the query cover seems to be skewed to only one part of the previous matches.		Answer: The e-values are lower this time but the query cover seems to be skewed to only one part of the previous matches.

Carol at 09:54, 6 November 2025

2025-11-06T09:54:36Z

← Older revision		Revision as of 11:54, 6 November 2025
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	* '''QUESTION 6''': Explore the the Graphic Summary tab. What can you say about the query coverage of the matches?		* '''QUESTION 6''': Explore the the Graphic Summary tab. What can you say about the query coverage of the matches?

			[[File:graphicSummary_PB2.png\|800px\|center]]

	Answer: Most query coverage of the hits is around 45-50%, however it seems that there are two regions of the protein that have separated hits, like if our orphan protein would contain a mix of two different proteins which seem to be abundant in many genus of bacteria.		Answer: Most query coverage of the hits is around 45-50%, however it seems that there are two regions of the protein that have separated hits, like if our orphan protein would contain a mix of two different proteins which seem to be abundant in many genus of bacteria.

Carol at 09:52, 6 November 2025

2025-11-06T09:52:52Z

← Older revision		Revision as of 11:52, 6 November 2025
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	NEW answers are being updated!		NEW answers are being updated!

	~~== When BLAST fails ==~~

	* '''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)?
	Answer: No sequences with E-value below 0.005.		Answer: No sequences with E-value below 0.005.


	~~==Trying another approach==~~

	[[File:question2_answer.png\|800px\|center]]		[[File:question2_answer.png\|800px\|center]]
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	* '''QUESTION 4''': Based on the first result, is there a clear homologue in non‐human species? What does that suggest about the gene’s taxonomic distribution?		* '''QUESTION 4''': Based on the first result, is there a clear homologue in non‐human species? What does that suggest about the gene’s taxonomic distribution?
	Apart from the orphan protein hit to itself, none of the hits are significant (E-values are in between 1-10, meaning the chance to get a random hit with the same score is one to ten sequences. The fact that the sequences are from Bacteria does not make the homology hypethesis very promising either.. but since a google search of the C22orf45 orphan gene suggests that the function is unknown we will continue the searches to see what we get.		Apart from the orphan protein hit to itself, none of the hits are significant (E-values are in between 1-10, meaning the chance to get a random hit with the same score is one to ten sequences. The fact that the sequences are from Bacteria does not make the homology hypethesis very promising either.. but since a google search of the C22orf45 orphan gene suggests that the function is unknown we will continue the searches to see what we get.



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

	* '''QUESTION 5''': After iteration 2, How many significant hits (E-value < 0.005) are now found? What happened with E-value of the hits found before?		* '''QUESTION 5''': After iteration 2, How many significant hits (E-value < 0.005) are now found? What happened with E-value of the hits found before?
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	Answer: During the first iteration a generic Blosum62 substitution matrix was used. The hits found there were made into a multiple alignment and a new and more sensitive position-specific-substitution-matrix (PSSM) based on the selected sequences, was constructed for the second iteration. This is why more sequences are found after the second iteration. A PSSM can capture evolutionary sequence information i.e. conserved regions, active sites and regions with less evolutionary pressure (many different amino acids at a certain position).		Answer: During the first iteration a generic Blosum62 substitution matrix was used. The hits found there were made into a multiple alignment and a new and more sensitive position-specific-substitution-matrix (PSSM) based on the selected sequences, was constructed for the second iteration. This is why more sequences are found after the second iteration. A PSSM can capture evolutionary sequence information i.e. conserved regions, active sites and regions with less evolutionary pressure (many different amino acids at a certain position).

	~~=== Saving and reusing the PSSM ===~~
	* '''QUESTION 8''': Can you see any changes on the results now? Look at the E-values, and the query cover on the Graphic Summary tab.		* '''QUESTION 8''': Can you see any changes on the results now? Look at the E-values, and the query cover on the Graphic Summary tab.
	Answer: The e-values are lower this time but the query cover seems to be skewed to only one part of the previous matches.		Answer: The e-values are lower this time but the query cover seems to be skewed to only one part of the previous matches.

Carol at 09:51, 6 November 2025

2025-11-06T09:51:16Z

@@ Line 47: / Line 47: @@
 * '''QUESTION 12''': What is the function of these proteins?
- >4A8E_A Chain A, The Structure Of A Dimeric Xer Recombinase From Archaea
+== Finding a remote homolog (on your own) ==
+* '''QUESTION 14''': Do you find any significant (E<0.005) hits? What is the E-value of the best hit?
 Answer: There are no significant hits. The best hit has an E-value of 6.9, and it is a hypothetical protein.
-* '''QUESTION 14''': How many significant (E<0.005) hits do you find now? What is the E-value of the best hit?
+* '''QUESTION 15''': How many significant (E<0.005) hits do you find now? What is the E-value of the best hit?
 Answer: There are 2 significant hits:
 * "GPI transamidase component Gaa1" from ''Trypanosoma melophagium'' with an E-value of 1e-05
 * "putative GPI transamidase component GAA1" from ''Trypanosoma theileri'' withs an E-value of 8e-04

Carol: /* Saving and reusing the PSSM */

2025-11-06T09:49:00Z

Saving and reusing the PSSM

@@ Line 34: / Line 34: @@
 === Saving and reusing the PSSM ===
-* '''QUESTION 8''': Do you find any significant PDB hits now? If yes, how many?
+* '''QUESTION 8''': Can you see any changes on the results now? Look at the E-values, and the query cover on the Graphic Summary tab.
-Answer: Yes, 13
+Answer: The e-values are lower this time but the query cover seems to be skewed to only one part of the previous matches.
-* '''QUESTION 9''': What are the PDB identifiers and the E-values for the two best PDB hits?
+* '''QUESTION 9''': Are there any homologous sequences found in search 2 that have an annotated function?
-Answer: 4A8E_A with an E-value of 2&times;10<sup>-19</sup>, 5HXY_A with an E-value of 8&times;10<sup>-19</sup>,
+In the previous search (PSI-BLAST run 2) the functions were mostly deaminase domain-containing protein and Rrf2 family transcriptional regulator and some hypethical proteins with unknown function.
-* '''QUESTION 10''': What are the values for Query coverage, sequence identity, and sequence similarity (Positives) for the two best PDB hits?
+* '''QUESTION 10''': Are there any homologous sequences found in search 3 that have an annotated function? Is there anything in common with search 2?
-'''Alignments:'''
+* '''QUESTION 11''': Do you find any significant PDB hits now? Look at the Graphic Summary and query coverage. Is this what you expected, Why?
- >4A8E_A Chain A, The Structure Of A Dimeric Xer Recombinase From Archaea
+* '''QUESTION 12''': What is the function of these proteins?
 === One more round ===

Carol: /* Constructing the PSSM */

2025-11-06T09:35:59Z

Constructing the PSSM

← Older revision		Revision as of 11:35, 6 November 2025
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	=== Constructing the PSSM ===		=== Constructing the PSSM ===
	* '''QUESTION 5''': How many significant hits does BLAST find (E-value < 0.005)?
	~~Answer: 500 (actually, much more than 500 hits, but BLAST by default only shows 500 — note that the last hit has an E-value much much smaller than 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''': After iteration 2, How many significant hits (E-value < 0.005) are now found? What happened with E-value of the hits found before?
	Answer: ~~approx~~. 50-~~60% sequence coverage, except one (#2) that~~ is ~~84%~~.		Answer: 500 (actually, much more than 500 hits, but BLAST by default only shows 500 — note that the last hit has an E-value much much smaller than 0.005). The E-values of the previous hits are much lower and look significant this time. This is because those sequences were integrated on the PSSM and therefore on the search.

	* '''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 6''': Explore the the Graphic Summary tab. What can you say about the query coverage of the matches?
	Answer: During the first iteration a generic Blosum62 substitution matrix was used~~, and~~ hits found there were made into a multiple alignment and ~~next~~ a more sensitive position-specific-substitution-matrix (PSSM). This is why more sequences are found in the second iteration. A PSSM can capture evolutionary sequence information i.e. conserved regions, active sites and regions with less evolutionary pressure (many different amino acids at a certain position).		Answer: Most query coverage of the hits is around 45-50%, however it seems that there are two regions of the protein that have separated hits, like if our orphan protein would contain a mix of two different proteins which seem to be abundant in many genus of bacteria.

			* '''QUESTION 7''': Why does BLAST come up with more significant hits in the second iteration? Explain in your own words the principle of profile‐based search in PSI-BLAST.
			Answer: During the first iteration a generic Blosum62 substitution matrix was used. The hits found there were made into a multiple alignment and a new and more sensitive position-specific-substitution-matrix (PSSM) based on the selected sequences, was constructed for the second iteration. This is why more sequences are found after the second iteration. A PSSM can capture evolutionary sequence information i.e. conserved regions, active sites and regions with less evolutionary pressure (many different amino acids at a certain position).

	=== Saving and reusing the PSSM ===		=== Saving and reusing the PSSM ===