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
Approaches to Atomic Resolution Structural Biology
NMR Spectroscopy X-ray Crystallography
ComputationDetermine experimentally or model
3D structures of biomolecules
*Use Cryo-EM, ESR, Fluorescence to build large structures from smaller pieces*
W. Chazin © 2003
High Resolution Structural Biology
Determine atomic structureAnalyze why molecules interact
W. Chazin © 2003
Anti-tumor activityDuocarmycin SA
The Reward: UnderstandingControl
Shape
Atomic interactions
W. Chazin © 2003
Object
Image plane
Eyepiece lensMagnification (N)
Objective lensMagnification (M)Diffracted light is recombined by this lens to forman image
Enlarged image of objectMagnification mn
Visible lightX-rays
Computer
Crystallographer
Electrondensity map
Model
Scattered rays
Detector
Object
Microscopy versus X-ray diffraction
Sylvie Doublié © 2000
Exploring 3D Molecular Structures Using NCBI Tools
A Field Guide
June 17, 2004
Structural Informatics
ChemicalFormula
3D Conformation
Function
ARKLMPQSCSW…ModificationsIonsLigands
Binding Sites Catalytic ResiduesKinetics ThermodynamicsSubstrates Intermediates
StructureDynamicsActive StatesFolding
Solving StructuresX-Ray Crystallography
Bond r (Å)
C-S 1.82
C-C 1.54
C-N 1.47
C-O 1.43
S-H 1.34
C=O 1.20
C-H 1.09
N-H 1.01
O-H 0.96
Electron Density Map
P F I
Resolution
5 Å 3 Å 1 Å
PDB
PDB File: HeaderHEADER ISOMERASE/DNA 01-MAR-00 1EJ9TITLE 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 ...
PDB File: DataATOM 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
AtomNumber
AtomName
ResidueName
Chain ID
ResidueNumber
YX Z
Occupancy
TemperatureFactor
Issues:Justification
Nomenclature
ATOM 1 N TRP A 203 30.156 -4.908 37.767 1.00 50.81
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/
Viewing Structures
C or CA Ball-and-stick CPK
Disulfide bond
Ribbon and Topology DiagramsRepresentations of Secondary Structures
-helix
-strand
N
C
GRASPGraphical Representation and Analysis
of Structural Properties
Red = negative surface chargeBlue = positive surface charge
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
Decoding the active site…
• How can we do this?
Biochemically
site-directed mutagenesis, three residues critical for enzyme activity have been identified: serine 80, aspartic acid 208, and histidine 236
7yas
