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The Genomics Knowledge You Need, When You Need It www.openhelix.com Exercises for the RCSB Protein Data Bank (Version 5, 2009) You can choose to do these using the questions as your only guide—or see the following pages for the step-by-step checklist to finding these answers. 1) Find out which structure the PDB ID “1cdw” denotes, where this structure was first published and what the molecule does. What is the molecular composition of this structure? Which functional and structural classifications does it belong to? What host species was used to clone this gene? Look at some images of this structure. Skills: Basic Search by PDB ID Number; Simple Information Gathering; Understanding the Structure Summary Page; Basic Visualization Techniques. 2) How many structures of TATA-Box Binding Proteins have been resolved from humans only (hint: use Boolean Operators)? Conduct a second search to look for TBPs that have been resolved from species other than humans. How many did you find and what was the range of species represented? Go to the structure summary page of Methanococcus jannaschii TBP (2Z8U) and find details of the crystallization experiments, including the diffraction source. Skills: Keyword Search with Boolean Operators; Retrieving Data; Understanding Results; Structure Summary Page Reports. 3) Were there many TBP protein structures deposited after January 1996 (hint: use Advanced Searching options)? Look for the human structure which has the highest resolution. How many of each type of secondary structures does this protein have? Which class of proteins is TBP a member of? Customize the structure summary page so that the derived data section is at the top. Skills: Advanced Searching; Understanding & Sorting Results; Using Links; Secondary Structure Information; Protein Classifications; Customizing the Structure Summary Page. RCSB Protein Data Bank Exercises, version 5. Correspond to the data available in January 2010 The materials and slides offered are for non-commercial use only. Reproduction, distribution and/or use for commercial purposes are strictly prohibited. Copyright 2009, OpenHelix, LLC. Seattle th 12600 SE 38 Street, Suite 230 Bellevue, WA 98006 (425) 401-1400 Boston 65 Main Street Somerville, MA 02145 (617) 627-9398 San Francisco 193 Haight Street San Francisco, CA 94012 (415) 252-1519 The Genomics Knowledge You Need, When You Need It www.openhelix.com Step-by-Step checklist/instructions for the RCSB Protein Data Bank introduction exercises 1) Find out which structure the PDB ID “1cdw” denotes, where this structure was first published and what the molecule does. What is the molecular composition of this structure? Which functional and structural classifications does it belong to? What host species was used to clone this gene? Look at some images of this structure. Step Action 1 2 3 4 5 6 7 8 9 Go to www.pdb.org, locate the Search query box on the top navigation bar. Leave the default search type as “PDB ID or keyword” and type in the PDB ID 1cdw. Click on the Search button. Note that you can also hit ‘enter’ on your keyboard. On the Structure Summary page that you are taken to, look at the title. Note that this structure is Human TBP (TATA-Box Binding Protein) core domain complexed with DNA. Find the primary citation to learn where and when this structure was first published and to get links directly to the PubMed abstract and PubMed Central full text article. Note that the structure was published by Nikolov et al., in the Proceedings of the National Academy of Science in 1996. Scroll down to the Molecular Description section to find out how many molecules are in this structure and what they are. Note that there are 2 DNA molecules in this structure (16-base-pair pieces of DNA that contain a TATA box) and a 179 amino acid polypeptide (the TATA Binding Protein, or TBP). Locate the Classification information at the top of the Molecular Description section to find out what the protein component of this structure does, in broad functional terms. Note that this protein is involved in DNA Transcription. Read the Source section to find the species that this protein is normally found in and the expression system host species. Note that it is a human protein and that E. coli was used as an expression system. You may also want to explore the following Derived Data section to learn about SCOP, CATH and PFAM classifications, and associated GO terminology. Click on “View in Jmol”, underneath the image on the top right hand of the Structure Summary page. Rotate the interactive image and examine it to see how clearly you can differentiate between the DNA and protein components. Note that right clicking on the interactive image gives viewing options. Notes: 2 The Genomics Knowledge You Need, When You Need It www.openhelix.com 2) How many structures of TATA-Box Binding Proteins have been resolved from humans only (hint: use Boolean Operators)? Conduct a second search to look for TBPs that have been resolved from species other than humans. How many did you find and what was the range of species represented? Go to the structure summary page of Methanococcus jannaschii TBP (2Z8U) and find details of the crystallization experiments, including the diffraction source. Step Action 1 2 3 4 5 6 7 8 9 Go to www.pdb.org, and locate the Search query box on the top navigation bar. Leave the default search type as “PDB ID or keyword” and type TBP AND human in the Search query box. Note that Boolean search combinations can be achieved using AND, OR and NOT as well as a few other terms. Also note that you do not need to use quotes for these terms. Click on the Search button. Note that a results page will open with several hits shown. Look at the top result tabs to see how many hits you found in various categories. Scroll down to read some of the titles in your structure results category (this is shown by default). Note that we found 15 TBP protein structures currently, but that will certainly change over time. Some of these are not human (like 1NH2) because the structure was annotated with the word “human” some place, and so it was picked up by this search. Return back to the top query box, type in TBP NOT human and click on the Search button. Note that you can also select the “Modify/Refine Query” from the upper right “Query Options” menu to be taken to the Advanced Search query form that you could also use to perform this action. Look at the top result tabs to see many hits you got. Scroll down to see the range of species by browsing through the titles. Note that you have found many more structures with this query. Note the wide range of species, including yeast and Drosophila. Click on entry 2Z8U to go to the structure summary page of Methanococcus jannaschii TPB. Select the Methods report tab at the top of the summary page. Note that you can also find some experimental details in the bottom right Experimental Details section on the structure summary page, but the Materials and Methods page provides many more. Scroll down to the Diffraction section and read through it. Note that the diffraction source is synchrotron. You can mouse over any of the headers to be taken to a definition and also utilize the top right options to display, download, print or share this file. Notes: 3 The Genomics Knowledge You Need, When You Need It www.openhelix.com 3) Were there many TBP protein structures deposited after January 1996 (hint: use Advanced Searching options)? Look for the human structure which has the highest resolution. How many of each type of secondary structures does this protein have? Which class of proteins is TBP a member of? Customize the structure summary page so that the derived data section is at the top. Step Action 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Go to www.pdb.org, and click on the Advanced Search link in the left navigation bar in the Search category. You can also access the Advanced Search from the top right link. From the Choose a Query Type dropdown menu, find the Structure Annotation section and choose a Structure Title query. Select Contains from the first dropdown menu and Type TBP in the query box to the right of it. Click on the Add Search Criteria to open a second query box. From the Choose a Query Type dropdown menu, choose the Deposit Date from the Deposition section. Enter 1996-01-01 for the first date, and for the second date select today’s date from the calendar by clicking on the box next to the query input box, and then clicking on the box highlighted with today’s date. Click on Submit Query. Note that currently there are more than 40 results shown. Click to open the Sort by menu on the top right of the page, and select the Resolution option. Scroll through the list of hits you obtained and look at the resolution of the crystals in Angstroms. Note that the list of results is resorted from lowest Angstroms to highest. Click on the human TBP PDB ID with the highest resolution (lowest Angstroms), for example select 1QNA at 1.80 A. Click on the Sequence tab at the top of its structure summary page, and look at the secondary structure information. Count how many alpha helices and extended strands there are. Note that you see 4 alpha helices and 12 beta strands and you can mouse over them to get a description. Now go back to the Structure Summary page either by clicking on the tab or the back button of your browser. Towards the bottom of the Structure Summary page, open the SCOP classifications in the Derived Data section to get information on the family, superfamily, fold and class of this protein. Note that TBP is a member of the alpha-beta proteins class. Click and drag the Derived Data section to the top of the page. Note that your page layout preferences will be saved until you select Reset Layout at the bottom of the page to return to default. You can also customize the homepage in a similar fashion. 4