1. The Strategy of Atomic Resolution Structural Biology Break down complexity so that the system can be understood at a fundamental level Build up a picture of the whole from the reconstruction of the high resolution pieces Understanding basic governing principles enables prediction, design, control Pharmaceuticals, biotechnology W. Chazin © 2003

2. Approaches to Atomic Resolution Structural Biology NMR Spectroscopy X-ray Crystallography Computation Determine experimentally or model 3D structures of biomolecules *Use Cryo-EM, ESR, Fluorescence to build large structures from smaller pieces* W. Chazin © 2003

3. High Resolution Structural Biology Determine atomic structure Analyze why molecules interact W. Chazin © 2003

4. Anti-tumor activity Duocarmycin SA The Reward: Understanding  Control Shape Atomic interactions W. Chazin © 2003

5. Object Image plane Eyepiece lens Magnification (N) Objective lens Magnification (M) Diffracted light is recombined by this lens to form an image Enlarged image of object Magnification mn Visible light X-rays Computer Crystallographer Electron density map Model Scattered rays Detector Object Microscopy versus X-ray diffraction Sylvie Doublié © 2000

6. Exploring 3D Molecular Structures Using NCBI Tools A Field Guide June 17, 2004

7. Structural Informatics Chemical Formula 3D Conformation Function ARKLMPQSCSW… Modifications Ions Ligands Binding SitesCatalytic Residues KineticsThermodynamics SubstratesIntermediates Structure Dynamics Active States Folding

8. Solving Structures X-Ray Crystallography Bondr (Å) C-S1.82 C-C1.54 C-N1.47 C-O1.43 S-H1.34 C=O1.20 C-H1.09 N-H1.01 O-H0.96 Electron Density Map P F I Resolution 5 Å 3 Å1 Å

9. PDB

10. PDB File: Header HEADER ISOMERASE/DNA 01-MAR-00 1EJ9 TITLE CRYSTAL STRUCTURE OF HUMAN TOPOISOMERASE I DNA COMPLEX COMPND MOL_ID: 1; COMPND 2 MOLECULE: DNA TOPOISOMERASE I; COMPND 3 CHAIN: A; COMPND 4 FRAGMENT: C-TERMINAL DOMAIN, RESIDUES 203-765; COMPND 5 EC: 5.99.1.2; COMPND 6 ENGINEERED: YES; COMPND 7 MUTATION: YES; COMPND 8 MOL_ID: 2; COMPND 9 MOLECULE: DNA (5'- COMPND 10 D(*C*AP*AP*AP*AP*AP*GP*AP*CP*TP*CP*AP*GP*AP*AP*AP*AP*AP*TP* COMPND 11 TP*TP*TP*T)-3'); COMPND 12 CHAIN: C; COMPND 13 ENGINEERED: YES; COMPND 14 MOL_ID: 3; COMPND 15 MOLECULE: DNA (5'- COMPND 16 D(*C*AP*AP*AP*AP*AP*TP*TP*TP*TP*TP*CP*TP*GP*AP*GP*TP*CP*TP* COMPND 17 TP*TP*TP*T)-3'); COMPND 18 CHAIN: D; COMPND 19 ENGINEERED: YES SOURCE MOL_ID: 1; SOURCE 2 ORGANISM_SCIENTIFIC: HOMO SAPIENS; SOURCE 3 EXPRESSION_SYSTEM_COMMON: BACULOVIRUS EXPRESSION SYSTEM; SOURCE 4 EXPRESSION_SYSTEM_CELL: SF9 INSECT CELLS; SOURCE 5 MOL_ID: 2; SOURCE 6 SYNTHETIC: YES; SOURCE 7 MOL_ID: 3; SOURCE 8 SYNTHETIC: YES KEYWDS PROTEIN-DNA COMPLEX, TYPE I TOPOISOMERASE, HUMAN REMARK 1 REMARK 2 REMARK 2 RESOLUTION. 2.60 ANGSTROMS. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : X-PLOR 3.1 REMARK 3 AUTHORS : BRUNGER … REMARK 280 REMARK 280 CRYSTALLIZATION CONDITIONS: 27% PEG 400, 145 MM MGCL2, 20 REMARK 280 MM MES PH 6.8, 5 MM TRIS PH 8.0, 30 MM DTT REMARK 290...

11. PDB File: Data ATOM 1 N TRP A 203 30.156 -4.908 37.767 1.00 50.81 N ATOM 2 CA TRP A 203 30.797 -4.667 36.431 1.00 49.96 C ATOM 3 C TRP A 203 30.369 -3.337 35.766 1.00 49.18 C ATOM 4 O TRP A 203 29.315 -3.238 35.147 1.00 49.27 O ATOM 5 CB TRP A 203 30.518 -5.863 35.513 1.00 46.77 C ATOM 6 CG TRP A 203 30.847 -5.651 34.081 1.00 44.60 C ATOM 7 CD1 TRP A 203 32.028 -5.234 33.553 1.00 49.72 C ATOM 8 CD2 TRP A 203 29.980 -5.876 32.984 1.00 43.73 C ATOM 9 NE1 TRP A 203 31.956 -5.191 32.177 1.00 45.45 N ATOM 10 CE2 TRP A 203 30.704 -5.582 31.805 1.00 45.23 C ATOM 11 CE3 TRP A 203 28.657 -6.305 32.877 1.00 46.48 C ATOM 12 CZ2 TRP A 203 30.149 -5.705 30.539 1.00 46.06 C ATOM 13 CZ3 TRP A 203 28.101 -6.431 31.622 1.00 43.08 C ATOM 14 CH2 TRP A 203 28.849 -6.131 30.463 1.00 45.77 C … Name Atom Number Atom Name Residue Name Chain ID Residue Number YXZ Occupancy Temperature Factor Issues: Justification Nomenclature ATOM 1 N TRP A 203 30.156 -4.908 37.767 1.00 50.81

12. Tools for Viewing Structures Protein Explorer –http://www.proteinexplorer.org Swiss PDB viewer –http://www.expasy.ch/spdbv PyMOL –http://pymol.sourceforge.net Kinemage –http://kinemage.biochem.duke.edu Rasmol –http://www.umass.edu/microbio/rasmol/

13. Viewing Structures C  or CA Ball-and-stickCPK Disulfide bond

14. Ribbon and Topology Diagrams Representations of Secondary Structures  -helix  -strand N C

15. GRASP Graphical Representation and Analysis of Structural Properties Red = negative surface charge Blue = positive surface charge

16. Let’s find cassava structures… http://www.pdb.org 1E89

17. Hydroxynitrile Lyase (EC 4.1.2.37) conversion of acetone cyanohydrin (the deglycosylation product of linamarin) to cyanide plus acetone process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by hydroxynitrile lyase

18. Decoding the active site… How can we do this?

19. Biochemically site-directed mutagenesis, three residues critical for enzyme activity have been identified: serine 80, aspartic acid 208, and histidine 236 7yas