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tutorials:workshop [2017/10/24 08:57] – Add structure scan picture and result link sayonitutorials:workshop [2019/06/19 15:02] (current) – sillitoe
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 **You are going to look at how the CATH database can help us in identifying the function of a particular protein structure.** **You are going to look at how the CATH database can help us in identifying the function of a particular protein structure.**
  
-The PDB structure, 4i6g, is an X-ray crystallography-solved structure for which the function has yet to be determined. However, this can be inferred by comparing the protein with other proteins of known function.  You can, for example, use the CATHEDRAL server to find a structural match in CATH. The CATHEDRAL server uses a structural comparison algorithm to compare a protein of interest (otherwise known as the 'query structure') against domains already classified in the CATH database. This means you can try to identify an unknown protein by comparing it with all known structures in CATH. +The PDB structure, 4i6g, is an X-ray crystallography-solved structure for which the function has yet to be determined. However, this can be inferred by comparing the protein with other proteins of known function.  You can, for example, use the CATHEDRAL server to find a structural match in CATH. The **CATHEDRAL** server uses a structural comparison algorithm to compare a protein of interest (otherwise known as the 'query structure') against domains already classified in the CATH database. This means you can try to identify an unknown protein by comparing it with all known structures in CATH. 
  
 The CATHEDRAL server can be found **[[http://www.cathdb.info/search/by_structure|here]]**. Please click the link. This will take you to a page that looks like this:  The CATHEDRAL server can be found **[[http://www.cathdb.info/search/by_structure|here]]**. Please click the link. This will take you to a page that looks like this: 
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 {{ :tutorials:4ig6_uploaded_to_cath.png }} {{ :tutorials:4ig6_uploaded_to_cath.png }}
  
-Each chain of the PDB can be submitted for structural scans separately. Submit **chain A** of the uploaded PDB to the structural scan by clicking on 'Submit Structure' for chain A. If the servers are busy, you might find that the job takes a long time to complete - you can skip the wait and view the previously calculated results **[[http://www.cathdb.info/search/grid_submission/9574|here]]**.+Each chain of the PDB can be submitted for structural scans separately. Submit **chain A** of the uploaded PDB to the structural scan by clicking on 'Submit Structure' for chain A. If the servers are busy, you might find that the job takes a long time to complete - you can skip the wait and view the previously calculated results **[[http://www.cathdb.info/search/grid_submission/12264|here]]**.
  
 A total of 528 matching structures in CATH v4.1 have been found, with scores ranging from very good (in green) through to very poor (in red). A total of 528 matching structures in CATH v4.1 have been found, with scores ranging from very good (in green) through to very poor (in red).
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 If you wish to explore other structural domains within a given S35 cluster, clicking on 'Show related domains' will open up a window containing the domain list. Clicking on a given domain ID will take you to its domain page. The data in the list can also be downloaded using the 'Download' button at the bottom of the window. If you wish to explore other structural domains within a given S35 cluster, clicking on 'Show related domains' will open up a window containing the domain list. Clicking on a given domain ID will take you to its domain page. The data in the list can also be downloaded using the 'Download' button at the bottom of the window.
  
-Please also visit the link to PDBsum on the domain page. PDBsum is a resource that stores information about all the protein files deposited in the PDB to learn more about the structural and functional characteristics of these domains.+Please also visit the link to PDBsum on the domain page. **PDBsum** is a resource that stores information about all the protein files deposited in the PDB to learn more about the structural and functional characteristics of these domains.
  
 ==== The HUP Superfamily ==== ==== The HUP Superfamily ====
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 We are now going to look more closely at the CATH superfamily in which 1dnpA01 is classified. This is the HUP domain superfamily (CATH code 3.40.50.620), named after **H**igh-signature proteins, **U**spA, and **P**P-loop NTPases which all contain this domain ([[http://www.ncbi.nlm.nih.gov/pubmed/12012333|reference]]). This superfamily is known to be structurally and functionally diverse ([[http://www.cell.com/structure/abstract/S0969-2126%2810%2900356-4|reference]]).  Here, we give a brief tour of some of the information held about this superfamily as displayed by our newly re-designed webpages before demonstrating how the CATH website can help investigate this diversity. We are now going to look more closely at the CATH superfamily in which 1dnpA01 is classified. This is the HUP domain superfamily (CATH code 3.40.50.620), named after **H**igh-signature proteins, **U**spA, and **P**P-loop NTPases which all contain this domain ([[http://www.ncbi.nlm.nih.gov/pubmed/12012333|reference]]). This superfamily is known to be structurally and functionally diverse ([[http://www.cell.com/structure/abstract/S0969-2126%2810%2900356-4|reference]]).  Here, we give a brief tour of some of the information held about this superfamily as displayed by our newly re-designed webpages before demonstrating how the CATH website can help investigate this diversity.
  
-The CATH webpage for the HUP superfamily can be accessed [[http://www.cathdb.info/version/latest/superfamily/3.40.50.620|here]]. A screenshot is shown in the figure below. There are a number of sections, which have been numbered 1 to 10.+The CATH webpage for the HUP superfamily can be accessed **[[http://www.cathdb.info/version/latest/superfamily/3.40.50.620|here]]**. A screenshot is shown in the figure below. There are a number of sections, which have been numbered 1 to 10.
  
 {{:tutorials:sf-page.png|}} {{:tutorials:sf-page.png|}}
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 Section 1 is a menu that you click on to navigate the site. From here you can explore the structural and functional features of the superfamily, references associated with all the protein domains within that superfamily, access to functionally annotated structural alignments, MDA, and the taxonomy browser.  Section 1 is a menu that you click on to navigate the site. From here you can explore the structural and functional features of the superfamily, references associated with all the protein domains within that superfamily, access to functionally annotated structural alignments, MDA, and the taxonomy browser. 
  
-A concise summary for the superfamily in the form of some useful statistics can be seen in section 9. It gives information on, for example, the number of domains, structural clusters and functional terms. For the HUP superfamily, it can be seen that there are 970 domains, and that there are 112 unique EC numbers and 409 unique Gene Ontology (GO) terms associated with the superfamily (click [[https://en.wikipedia.org/wiki/Gene_ontology|here]] for more info on GO terms).+A concise summary for the superfamily in the form of some useful statistics can be seen in section 9. It gives information on, for example, the number of domains, structural clusters and functional terms. For the HUP superfamily, it can be seen that there are 970 domains and that there are 112 unique EC numbers and 409 unique Gene Ontology (GO) terms associated with the superfamily (click [[https://en.wikipedia.org/wiki/Gene_ontology|here]] for more info on GO terms).
  
-An indication of just how structurally diverse the HUP family is shown in section 6. Here, you can scroll though the smallest, largest and a representative structure (according to the number of residues) belonging to the HUP superfamily.+An indication of just how structurally diverse the HUP family is shown in section 6. Here, you can scroll through the smallest, largest and a representative structure (according to the number of residues) belonging to the HUP superfamily.
  
 The box below shows a 3D structural superposition between the smallest (2pfsA01) and largest domain (1wkbA01) displayed using the program Jmol. What you see initially is a wireframe representation of the superposition, which isn't very clear for this purpose, but if you press the grey button labeled 'Click here', the two domains will be coloured differently and the wireframe representation will be replaced by a cartoon representation of the structures, making it much easier to compare them. 2pfsA01 is coloured blue. For 1wkbA01, those parts of the structure that superimpose well with 2pfsA01 are coloured red and the rest of the structure, termed structural embellishments, are coloured pink. This superposition shows considerable embellishments in the larger structure compared to 2pfsA01, indicating the structural diversity between these two relatives.  The box below shows a 3D structural superposition between the smallest (2pfsA01) and largest domain (1wkbA01) displayed using the program Jmol. What you see initially is a wireframe representation of the superposition, which isn't very clear for this purpose, but if you press the grey button labeled 'Click here', the two domains will be coloured differently and the wireframe representation will be replaced by a cartoon representation of the structures, making it much easier to compare them. 2pfsA01 is coloured blue. For 1wkbA01, those parts of the structure that superimpose well with 2pfsA01 are coloured red and the rest of the structure, termed structural embellishments, are coloured pink. This superposition shows considerable embellishments in the larger structure compared to 2pfsA01, indicating the structural diversity between these two relatives. 
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 === Investigating the Structural and Functional diversity within the HUP Superfamily using CATH === === Investigating the Structural and Functional diversity within the HUP Superfamily using CATH ===
  
-This brings us into the next part of this tutorial in which we are going to explore the structural and functional diversity of the HUP superfamily using CATH. The structure and function of a protein is closely linked, so it is natural to assume that structural diversity is likely to result in functional diversity within a superfamily. Sections 2 and 3 on the homepage (see below) gives information on the functional diversity and lets you view the distribution of GO annotations and EC numbers associated with this superfamily. Placing your mouse over one of the pie segments gives the EC number or GO term, the name of that function and also the incidence of the functional annotation in question within the superfamily as a percentage. There are currently a total of 1159 GO annotations and 149 EC annotations for the HUP superfamily.+This brings us to the next part of this tutorial in which we are going to explore the structural and functional diversity of the HUP superfamily using CATH. The structure and function of a protein are closely linked, so it is natural to assume that structural diversity is likely to result in functional diversity within a superfamily. Sections 2 and 3 on the homepage (see below) gives information on the functional diversity and lets you view the distribution of GO annotations and EC numbers associated with this superfamily. Placing your mouse over one of the pie segments gives the EC number or GO term, the name of that function and also the incidence of the functional annotation in question within the superfamily as a percentage. There are currently a total of 1159 GO annotations and 149 EC annotations for the HUP superfamily.
  
 {{tutorials:go-ec.png?maxwidth=400}} {{tutorials:go-ec.png?maxwidth=400}}
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 The HUP superfamily is known to be particularly functionally diverse. Here, we concentrate our efforts on looking at two domains  [[http://www.cathdb.info/version/latest/domain/1od6A00|1od6A00]] (EC 2.7.7.3) and [[http://www.cathdb.info/version/latest/domain/1f7uA01|1f7uA01]] (EC 6.1.1.19).  The HUP superfamily is known to be particularly functionally diverse. Here, we concentrate our efforts on looking at two domains  [[http://www.cathdb.info/version/latest/domain/1od6A00|1od6A00]] (EC 2.7.7.3) and [[http://www.cathdb.info/version/latest/domain/1f7uA01|1f7uA01]] (EC 6.1.1.19). 
  
-[[http://www.ebi.ac.uk/thornton-srv/databases/MACiE/|MACiE]] is a database maintained though a collaboration between the Thornton group at the European Bioinformatics Institute and the Mitchell Group at the University of St Andrew, and it stores enzyme reaction mechanisms. It can be searched by the Catalytic Domain CATH Code (in this case, 3.40.50.620). If you type in the CATH code in the field adjacent to the 'Search Catalytic Domain CATH code' button and then click, a page will be displayed providing all the general information held for the HUP superfamily. 12 different reaction mechanisms are recorded in MACiE for this superfamily.  There are many relatives having different enzyme classification numbers at the third level (EC3) in this family, which is suggestive of changes in chemistry between some relatives within this superfamily (see figure below).+**[[http://www.ebi.ac.uk/thornton-srv/databases/MACiE/|MACiE]]** is a database maintained through a collaboration between the Thornton group at the European Bioinformatics Institute and the Mitchell Group at the University of St Andrew, and it stores enzyme reaction mechanisms. It can be searched by the Catalytic Domain CATH Code (in this case, 3.40.50.620). If you type in the CATH code in the field adjacent to the 'Search Catalytic Domain CATH code' button and then click, a page will be displayed providing all the general information held for the HUP superfamily. 12 different reaction mechanisms are recorded in MACiE for this superfamily.  There are many relatives having different enzyme classification numbers at the third level (EC3) in this family, which is suggestive of changes in chemistry between some relatives within this superfamily (see figure below).
  
 {{tutorials:macie.png}} {{tutorials:macie.png}}
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 // //
  
-There is a link at the bottom of the page to an overview to all MACiE results for this superfamily labelled **OVERVIEW OF ALL RESULTS** which you can go through to explore the extent of the different enzyme reaction mechanisms present. It brings up a page where you can find more information on EC number distribution, CATH domain partners and information of the catalytic residues and cofactors present. Relatives in this superfamily have many different enzyme classifications at the third+There is a link at the bottom of the page to an overview of all MACiE results for this superfamily labelled **OVERVIEW OF ALL RESULTS** which you can go through to explore the extent of the different enzyme reaction mechanisms present. It brings up a page where you can find more information on EC number distribution, CATH domain partners and information of the catalytic residues and cofactors present. Relatives in this superfamily have many different enzyme classifications at the third
 level (i.e. different EC3 numbers), which is suggestive of changes in chemistry throughout this superfamily (click [[http://en.wikipedia.org/wiki/Enzyme_classification|here]] for more info on EC numbers). level (i.e. different EC3 numbers), which is suggestive of changes in chemistry throughout this superfamily (click [[http://en.wikipedia.org/wiki/Enzyme_classification|here]] for more info on EC numbers).
  
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 If you then go back to the list of MACiE entries and click on the entries for our example domains (M0299, Pantothenate synthetase, EC 6.3.2.1 for 1od6A00 and M0235, Arginyl-tRNA synthetase, EC 6.1.1.19 for 1f7uA01), you can see the overall reactions for these enzymes. It can be seen that both 1od6A00 and 1f7uA01 are ligases, but they have different substrates and form different products. If you then go back to the list of MACiE entries and click on the entries for our example domains (M0299, Pantothenate synthetase, EC 6.3.2.1 for 1od6A00 and M0235, Arginyl-tRNA synthetase, EC 6.1.1.19 for 1f7uA01), you can see the overall reactions for these enzymes. It can be seen that both 1od6A00 and 1f7uA01 are ligases, but they have different substrates and form different products.
    
-It is clear from these results that the HUP superfamily is associated with a significant number of different enzyme reaction mechanisms. There are a number of possible reasons for this functional diversity. To explore how these enzymes may have evolved different functions, we can look for structural changes within the family. Here, we compare the structures of our two HUP domain examples using our in-house structural comparison algorithm called SSAP. +It is clear from these results that the HUP superfamily is associated with a significant number of different enzyme reaction mechanisms. There are a number of possible reasons for this functional diversity. To explore how these enzymes may have evolved different functions, we can look for structural changes within the family. Here, we compare the structures of our two HUP domain examples using our in-house structural comparison algorithm called **SSAP**. 
  
 Whilst the CATHEDRAL algorithm you used at the beginning of the tutorial is fast and allows you to search all structures in CATH, SSAP is a slower and slightly more accurate method for comparing two protein structures.  Whilst the CATHEDRAL algorithm you used at the beginning of the tutorial is fast and allows you to search all structures in CATH, SSAP is a slower and slightly more accurate method for comparing two protein structures. 
  
-SSAP takes two structures and calculates how similar they are in structure, residue-by-residue. Similarity is measured by the SSAP score. This score ranges from 0 to 100; a score of 100 would indicate that the two structures were effectively identical. Please click [[http://cath-tools.cathdb.info/pairwise|here]] to go to the SSAP server page. Type in 1od6A00 as Domain ID 1 and 1f7uA01 as Domain ID 2. Press 'GO'.+SSAP takes two structures and calculates how similar they are in structure, residue-by-residue. Similarity is measured by the SSAP score. This score ranges from 0 to 100; a score of 100 would indicate that the two structures were effectively identical. Please click **[[http://cath-tools.cathdb.info/structure/pairwise|here]]** to go to the SSAP server page. Type in **1od6A00** as Domain ID 1 and **1f7uA01** as Domain ID 2. Press 'GO'.
  
 From this superposition we can see that the two domains are significantly different in structure. This structural divergence is also clearly highlighted by their SSAP score of 58.77 and an RMSD of 8.15Å. From this superposition we can see that the two domains are significantly different in structure. This structural divergence is also clearly highlighted by their SSAP score of 58.77 and an RMSD of 8.15Å.
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 The superposition shows that, although there is a structural core common to both structures, 1f7uA01 has some considerable structural embellishments not seen in 1od6A00. There are also noticeable shifts in the positions of the catalytic site residues.   The superposition shows that, although there is a structural core common to both structures, 1f7uA01 has some considerable structural embellishments not seen in 1od6A00. There are also noticeable shifts in the positions of the catalytic site residues.  
  
-2DSEC ([[http://www.sciencedirect.com/science/article/pii/S0022283606006176|reference]]) is an algorithm that provides a schematic representation of protein structural features. It employs a multiple structural alignment to create a summary of all the secondary structures present for each structure in the alignment. Circles represent alpha-helices and triangles a beta strand. The size of the circle or triangle is determined by the size of the secondary structure it is representing. Core secondary structure elements are represented as light pink circles and yellow triangles. Embellishments are coloured as dark pink circles and brown triangles.+**2DSEC** ([[http://www.sciencedirect.com/science/article/pii/S0022283606006176|reference]]) is an algorithm that provides a schematic representation of protein structural features. It employs a multiple structural alignment to create a summary of all the secondary structures present for each structure in the alignment. Circles represent alpha-helices and triangles a beta strand. The size of the circle or triangle is determined by the size of the secondary structure it is representing. Core secondary structure elements are represented as light pink circles and yellow triangles. Embellishments are coloured as dark pink circles and brown triangles.
  
 The 2DSEC plot for the HUP examples 1f7uA01 and 1od6A00 is shown below: The 2DSEC plot for the HUP examples 1f7uA01 and 1od6A00 is shown below:
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 The 2DSEC plot confirms the findings of the SSAP superposition; 1f7uA01 has some extensive structural embellishments, mainly alpha-helical regions, when compared to the smaller 1od6A00 structure.  The 2DSEC plot confirms the findings of the SSAP superposition; 1f7uA01 has some extensive structural embellishments, mainly alpha-helical regions, when compared to the smaller 1od6A00 structure. 
  
-Recruitment of different domain partners can also result in changes in protein function. There is a link to a third party application called Archschema ([[http://www.ncbi.nlm.nih.gov/pubmed/20299327?dopt=Abstract|reference]]) on the main superfamily home page (see section 7 on the homepage figure). This  generates dynamic plots of related Pfam multi-domain architectures (MDAs).  To get an overall view of the number of different, related Pfam architectures in this family, click on the link to boot up the application. You will get a graph of related CATH MDAs for this family. In order to view those architectures that are most likely to be accurate, select the **search** tag and then select reviewed UniProt sequences only. Press **refine search** and you will be presented with a plot showing 84 MDAs (see figure below):+Recruitment of different domain partners can also result in changes in protein function. There is a link to a third party application called **Archschema** ([[http://www.ncbi.nlm.nih.gov/pubmed/20299327?dopt=Abstract|reference]]) on the main superfamily home page (see section 7 on the homepage figure). This generates dynamic plots of related Pfam multi-domain architectures (MDAs).  To get an overall view of the number of different, related Pfam architectures in this family, click on the link to boot up the application. You will get a graph of related CATH MDAs for this family. In order to view those architectures that are most likely to be accurate, select the **search** tag and then select reviewed UniProt sequences only. Press **refine search** and you will be presented with a plot showing 84 MDAs (see figure below):
  
 {{:tutorials:arch.png|}} {{:tutorials:arch.png|}}
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 Now that we have an idea of the scale of the number of domain partners associated with the family as a whole, we will now return to comparing our two HUP examples using a different resource. Gene3D assigns CATH domains to genes and annotates them with functional and structural information. We are going to use Gene3D to compare the MDAs of our examples. Multi-chain architectures show all the domains contained within a protein chain. Now that we have an idea of the scale of the number of domain partners associated with the family as a whole, we will now return to comparing our two HUP examples using a different resource. Gene3D assigns CATH domains to genes and annotates them with functional and structural information. We are going to use Gene3D to compare the MDAs of our examples. Multi-chain architectures show all the domains contained within a protein chain.
  
- +Next, go to the Gene3D v14 website protein search page [[http://gene3d.biochem.ucl.ac.uk/searchForm?mode=protein|here]]. Input the PDB code, 1od6 into the search box and click the **Get Results** button. The resulting page will first summarise the list of domain families that are assigned to this query protein (which in this case is just a single protein chain). From the Summary section, and the Domain View section just below, we can see that a single domain has been identified within this protein, which has been assigned to a functional family named "Phosphopantetheine adenylyltransferase". Scrolling further down the page provides information associated with this query protein such as the: protein sequence, predicted GO term function annotations, known drug targets that bind to this protein, UniProt entries, and Ensembl entries.
- +
-Next go to the Gene3D v14 website protein search page [[http://gene3d.biochem.ucl.ac.uk/searchForm?mode=protein|here]]. Input the PDB code, 1od6 into the search box and click the **Get Results** button. The resulting page will first summarise the list of domain families that are assigned to this query protein (which in this case is just a single protein chain). From the Summary section, and the Domain View section just below, we can see that a single domain has been identified within this protein, which has been assigned to a functional family named "Phosphopantetheine adenylyltransferase". Scrolling further down the page provides information associated with this query protein such as the: protein sequence, predicted GO term function annotations, known drug targets that bind to this protein, UniProt entries, and Ensembl entries.+
  
  
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 Clicking on the 'OVERVIEW' link at the bottom of the page brings up a page providing more information on EC number distribution, CATH domain partners and information of the catalytic residues and cofactors present. Scrolling down takes you to a table of **Catalytic Machinery Similarities**. It compares pairs of catalytic mechanisms present in the Aldolases and calculates how similar they are using an algorithm that combines information on catalytic residues and superposition of the active site. The similarity score is between 0-1. The lower the score, the more different the reaction mechanisms. Clicking on the 'OVERVIEW' link at the bottom of the page brings up a page providing more information on EC number distribution, CATH domain partners and information of the catalytic residues and cofactors present. Scrolling down takes you to a table of **Catalytic Machinery Similarities**. It compares pairs of catalytic mechanisms present in the Aldolases and calculates how similar they are using an algorithm that combines information on catalytic residues and superposition of the active site. The similarity score is between 0-1. The lower the score, the more different the reaction mechanisms.
  
-For this tutorial, we are most interested in comparing the reaction mechanisms associated with two relatives having different functions. For example, 1h7oA00, Aminolevulinate dehydratase (EC 4.2.1.24) and 1d3gA00, Dihydroorotate oxidase (EC 1.3.3.1). Have a look for the reaction mechanisms corresponding to these ECs in the Catalytic Machinery Similarities table and draw your own conclusion. For more information on this comparison, click on the link within the table. This takes you to a page that compares the two reaction mechanisms side by side.+For this tutorial, we are most interested in comparing the reaction mechanisms associated with two relatives having different functions. For example, **1h7oA00**, Aminolevulinate dehydratase (EC 4.2.1.24) and **1d3gA00**, Dihydroorotate oxidase (EC 1.3.3.1). Have a look for the reaction mechanisms corresponding to these ECs in the Catalytic Machinery Similarities table and draw your own conclusion. For more information on this comparison, click on the link within the table. This takes you to a page that compares the two reaction mechanisms side by side.
  
 So, how are these changes in mechanisms mediated?  So, how are these changes in mechanisms mediated? 
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 Firstly, we can explore whether there are any significant structural differences between the domains associated with these functions.  Firstly, we can explore whether there are any significant structural differences between the domains associated with these functions. 
  
-Within a CATH superfamily, structurally-similar relatives are grouped into structural clusters. Each structural cluster is then clustered again into functional families, or FunFams (FFs). The clustering that produces our functional families is performed by our in-house protocol, FunFHMMer ([[http://bioinformatics.oxfordjournals.org/content/early/2015/07/29/bioinformatics.btv398.long| reference]]). Each domain clustered within a particular FunFam is predicted to have the same, or a very similar, protein function.+Within a CATH superfamily, structurally-similar relatives are grouped into structural clusters. Each structural cluster is then clustered again into functional families, or **FunFams** (FFs). The clustering that produces our functional families is performed by our in-house protocol, FunFHMMer ([[http://bioinformatics.oxfordjournals.org/content/early/2015/07/29/bioinformatics.btv398.long| reference]]). Each domain clustered within a particular FunFam is predicted to have the same, or a very similar, protein function.
  
 If we go back to the homepage for the 3.20.20.70 superfamily, you will see the functional families tree (see section 5 of the homepage - see below). The Aldolase Class I relatives are clustered into 19 structural clusters, all of which have one or more functional families. There are 286 functional families within this superfamily. If we go back to the homepage for the 3.20.20.70 superfamily, you will see the functional families tree (see section 5 of the homepage - see below). The Aldolase Class I relatives are clustered into 19 structural clusters, all of which have one or more functional families. There are 286 functional families within this superfamily.
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 The next thing we can look at is whether or not there are local changes, particularly around the active site, for example, residue mutations in the site and changes in catalytic residues. Taking 1h7oA00 and 1d3gA00 as our examples, we can go back to their respective functional family pages and look at the multiple alignments for those families. Highly conserved residues are highlighted in the alignment (as shown above) and the structure and you can compare them side by side to observe any differences. We are currently in the process of adding in catalytic residue information to the FunFam pages so that conserved residue and catalytic residue information can be viewed on the FunFam MSA and the representative structure. The next thing we can look at is whether or not there are local changes, particularly around the active site, for example, residue mutations in the site and changes in catalytic residues. Taking 1h7oA00 and 1d3gA00 as our examples, we can go back to their respective functional family pages and look at the multiple alignments for those families. Highly conserved residues are highlighted in the alignment (as shown above) and the structure and you can compare them side by side to observe any differences. We are currently in the process of adding in catalytic residue information to the FunFam pages so that conserved residue and catalytic residue information can be viewed on the FunFam MSA and the representative structure.
  
-We can also use SSAP to create a superposition of our two proteins and then compare the position of functional residues. Just type 1h7oA00 as protein 1 and 1d3gA00 as protein 2. An interactive RasMol image of the superimposed structures can be brought up by pressing the **Launch Rasmol** button. Initially, a simple backbone superposition will be displayed but you can change to a cartoon display by typing in **select*** and **cartoon on** in the command console (see picture below) +We can also use [[http://cath-tools.cathdb.info/structure/pairwise|SSAP]] to create a superposition of our two proteins and then compare the position of functional residues. Just type **1h7oA00** as protein 1 and **1d3gA00** as protein 2 and click on ‘GO’. An interactive LiteMol visualization of the superimposed structures in cartoon representations is shown.
- +
-{{:tutorials:rasmol.png|}}+
  
 +{{:tutorials:ssap_litemol.png|}}
  
 The [[http://www.ebi.ac.uk/thornton-srv/databases/CSA/| Catalytic Site Atlas]] is a database containing enzyme active sites and catalytic residues in enzymes. We want to use this resource to determine the catalytic residues for our aldolase examples and map them onto the RasMol 3D structure. At the top of the homepage, you will find a field labelled **PDB code**. Type in 1h7o and then 1d3g to get a list of catalytic residues for these proteins (see picture below for example) The [[http://www.ebi.ac.uk/thornton-srv/databases/CSA/| Catalytic Site Atlas]] is a database containing enzyme active sites and catalytic residues in enzymes. We want to use this resource to determine the catalytic residues for our aldolase examples and map them onto the RasMol 3D structure. At the top of the homepage, you will find a field labelled **PDB code**. Type in 1h7o and then 1d3g to get a list of catalytic residues for these proteins (see picture below for example)
  
-Once you have your catalytic residues, highlight them on your RasMol superposition using the following commands - **select n1, n2, n3** etc (where n//x// denotes a catalytic residue number, for example, 17) then **spacefill** and then select a color - for example type **colour purple** if you want the catalytic residues for one of the proteins to be purple. +A jmol of the SSAP superposition has been provided with the catalytic residues of the domains highlighted. Here, 1h7oA00 is in pink, with its catalytic residues red and 1d3gA00 light blue with its catalytic residues blue
- +
-A jmol of the SSAP superposition has been provided in case you have difficulties with the SSAP server. Here, 1h7oA00 is in pink, with its catalytic residues red and 1d3gA00 light blue with its catalytic residues blue+
  
 <jsmol 1h7o_2 :tutorials:1h7oA00_1d3gA00.pdb.gz 80% 400> <jsmol 1h7o_2 :tutorials:1h7oA00_1d3gA00.pdb.gz 80% 400>
Line 245: Line 240:
 </jsmol>  </jsmol> 
  
-It can clearly be seen that the catalytic residues of these two domains are in different 3D locations in the active site. An SSAP alignment of the two domains is below which highlights catalytic residues according to their properties. Aromatic residues are in red, polar residues in green and those with a positive charge are in purple.+It can clearly be seen that the catalytic residues of these two domains are in different 3D locations in the active site. A SSAP alignment of the two domains is below which highlights catalytic residues according to their properties. Aromatic residues are in red, polar residues in green and those with a positive charge are in purple.
  
 {{:tutorials:tut.png|}} {{:tutorials:tut.png|}}
tutorials/workshop.1508835466.txt.gz · Last modified: by sayoni

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