the strategy of atomic resolution structural biology break down complexity so that the system can be...

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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

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Page 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

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

Page 2: 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

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

Page 3: 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

High Resolution Structural Biology

Determine atomic structureAnalyze why molecules interact

W. Chazin © 2003

Page 4: 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

Anti-tumor activityDuocarmycin SA

The Reward: UnderstandingControl

Shape

Atomic interactions

W. Chazin © 2003

Page 5: 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

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

Page 6: 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

Exploring 3D Molecular Structures Using NCBI Tools

A Field Guide

June 17, 2004

Page 7: 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

Structural Informatics

ChemicalFormula

3D Conformation

Function

ARKLMPQSCSW…ModificationsIonsLigands

Binding Sites Catalytic ResiduesKinetics ThermodynamicsSubstrates Intermediates

StructureDynamicsActive StatesFolding

Page 8: 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

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

Page 9: 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

PDB

Page 10: 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

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 ...

Page 11: 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

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

Page 12: 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

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/

Page 13: 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

Viewing Structures

C or CA Ball-and-stick CPK

Disulfide bond

Page 14: 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

Ribbon and Topology DiagramsRepresentations of Secondary Structures

-helix

-strand

N

C

Page 15: 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

GRASPGraphical Representation and Analysis

of Structural Properties

Red = negative surface chargeBlue = positive surface charge

Page 16: 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

Let’s find cassava structures…

http://www.pdb.org

1E89

Page 17: 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

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

Page 18: 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

Decoding the active site…

• How can we do this?

Page 19: 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

Biochemically

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

7yas