1. Module 3 Protein Structure Database/Structure Analysis Learning objectives Understand how information is stored in PDB Learn how to read a PDB flat file Become familiar with comparative protein modelling theories Learn how to search the PDB for information. Learn how to download a structure Learn how to display a structure on the screen Learn how to compare two structures on the screen.

2. Primary, secondary, supersecondary, and tertiary structure Primary Secondary Supersecondary Tertiary ACFTYPL … ACFTYPL sssccss

3. ATOM 1 N ARG A 14 22.451 98.825 31.990 1.00 88.84 N ATOM 2 CA ARG A 14 21.713 100.102 31.828 1.00 90.39 C ATOM 3 C ARG A 14 22.583 101.018 30.979 1.00 89.86 C ATOM 4 O ARG A 14 22.105 101.989 30.391 1.00 89.82 O ATOM 5 CB ARG A 14 21.424 100.704 33.208 1.00 93.23 C ATOM 6 CG ARG A 14 20.465 101.880 33.215 1.00 95.72 C ATOM 7 CD ARG A 14 20.008 102.147 34.637 1.00 98.10 C ATOM 8 NE ARG A 14 18.999 103.196 34.718 1.00100.30 N ATOM 9 CZ ARG A 14 18.344 103.507 35.833 1.00100.29 C ATOM 10 NH1 ARG A 14 18.580 102.835 36.952 1.00 99.51 N ATOM 11 NH2 ARG A 14 17.441 104.479 35.827 1.00100.79 N Part of a record from the PDB X Y Z

4. What is PDB? Protein structure database Annotated records that represent three dimensional coordinates of atoms of biological molecules Generated from direct submissions of coordinates from the authors. In the old days, this was called the Brookhaven National Database.

5. 3D structure data The largest 3D structure database is the Protein Database It contains over 15,000 records Each record contains 3D coordinates for macromolecules 80% of the records were obtained from X-ray diffraction studies, 16% from NMR and the rest from other methods and theoretical calculations

6. Protein structure viewers RasMol Deep View Cn3D WebLabViewer

7. Steps to tertiary structure prediction Comparative protein modeling Extrapolates new structure based on related family members Steps 1. Identification of modeling templates 2. Alignment 3. Model building

8. Identification of modeling templates One chooses a cutoff value from FastA or BLAST search Up to ten templates can be used but the one with the highest sequence similarity is the reference template C  atoms are selected for superimposition

9. Alignment Optimization of superimposition of templates “Common core” and conserved loops of target sequence is threaded onto the template structure

10. Building the model Framework construction Average the position of each atom in target, based on the corresponding atoms in template. Areas that do not match the template are constructed by using a “spare part” algorithm Completing the backbone-a library of PDB entries is consulted Side chains are added Model refinement-minimization of energy

11. Framework construction

14. Alpha Helix

16. show all the groups within 8 A of N of NAG202

17. Comparing similar polypeptides One of the first structures solved was that of hemoglobin. It is composed of 2 alpha and 2 beta chains. The alpha and beta chains have similar structure. With Deep View you can superimpose the chains and locate the amino acids that are near one another in the two chains. We will also print out an alignment of the two polypeptides.

18. Superimposition of two polypeptides

19. Workshop for module 3: Download hen lysozyme structure file from PDB. Show the Ramachandran plot associated with this structure. Find out the residues that surround the active site of the enzyme. Download the human hemoglobin structure from PDB. Separate the alpha chain and beta chain into two separate files. Compare the structures. Print out the overlapped structures and print out the aligned primary sequences.