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tutorials:workshop [2017/10/24 09:11] – sayonitutorials:workshop [2019/06/19 15:02] (current) – sillitoe
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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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 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 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).+**[[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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 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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 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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 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>
tutorials/workshop.1508836301.txt.gz · Last modified: by sayoni

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