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Jan. 7, 2009. Biochemistry 300 Introduction to Structural Biology. Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@vanderbilt.edu http://structbio.vanderbilt.edu/chazin/classnotes/. Biology is Organized into Structures. Organ  Tissue  Cell  Molecule  Atoms.

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biochemistry 300 introduction to structural biology

Jan. 7, 2009

Biochemistry 300Introduction to Structural Biology

Walter Chazin

5140 BIOSCI/MRBIII

E-mail: Walter.Chazin@vanderbilt.edu

http://structbio.vanderbilt.edu/chazin/classnotes/

slide2

Biology is Organized into Structures

Organ  Tissue  Cell  Molecule  Atoms

  • A cell is an organization of millions of molecules
  • Proper communication between these molecules is essential to the normal functioning of the cell
  • To understand communication: *Determine the arrangement of atoms*
what is structural biology

3D

structure

Organism

Cell

What is Structural Biology?

Sequence

Structural Scales

MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIPREFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILIKENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASEMRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL

polymerase

SSBs

Complexes

helicase

primase

Assemblies

Cell

Structures

System Dynamics

slide4

Atomic Resolution Structural Biology

  • Determine atomic structure to analyze why molecules interact
slide5

Anti-tumor activity

Duocarmycin SA

Atomic interactions

The Reward: UnderstandingControl

Shape

breakdown strategy for atomic resolution structural biology
Breakdown Strategy for Atomic Resolution Structural Biology
  • Divide into domains 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 AND test model with intact proteins
  • Understanding basic governing principles enables prediction, design, control
slide8

P

Characterize Quaternary Structure

14/32D/70C

70AB

X-ray

Zn

B

A

C

D

RPA70

RPA32

RPA14

NTD

NMR

14

CTD

70NTD

32CTD

quaternary structure?

techniques for atomic resolution structural biology
Techniques for Atomic Resolution Structural Biology

NMR Spectroscopy X-ray Crystallography

Computation

Determine experimentally or model 3D structures of biomolecules

structure determined differently by x ray and nmr

X-ray

NMR

RF

Resonance

Diffraction

Pattern

X-rays

RF

H0

  • Direct detection of

atom positions

  • Crystals
  • Indirect detection via

H-H distances

  • In solution
Structure Determined Differently by X-ray and NMR
why structure in silico
Why Structure in silico?
  • A good guess is better than nothing!
    • Enables the design of experiments
    • Potential for high-throughput
  • Crystallography and NMR don’t always work!
    • Many important proteins do not crystallize
    • Size limitations with NMR
  • Invaluable for analyzing/understanding structure
computational approaches molecular simulations
Computational ApproachesMolecular Simulations
  • Convert experimental data into structures
  • Predict effects of mutations, changes in environment
  • Insight into molecular motions
  • Interpret structures- characterize the chemical properties (e.g. surface) to infer function
computational approaches structure prediction

1 QQYTA KIKGR

11 TFRNE KELRD

21 FIEKF KGR

Algorithm

Computational ApproachesStructure Prediction
  • Secondary structure (only sequence)
  • Homology modeling (using related structure)
  • Fold recognition
  • Ab-initio 3D prediction: “The Holy Grail”
complementarity of the atomic resolution methods
Complementarity of theAtomic Resolution Methods
  • X-ray crystallography- highest resolution structures; faster than NMR
  • NMR- enables widely varying solution conditions; characterization of motions and dynamic, weakly interacting systems
  • Computation- models without experiment; very fast; fundamental understanding of structure, dynamics and interactions (provides the why answers)
techniques for near atomic resolution structural biology
Techniques for Near-Atomic Resolution Structural Biology

NMR Spectroscopy X-ray Crystallography

Computation

  • Determine experimentally or model 3D structures of biomolecules
  • EPR/Fluorescence to measure distances when traditional methods fail
  • EM/Scattering to get snapshots of whole molecular structures
  • (Cryo-EM starts to approach atomic resolution!)
representation of structure conformational ensemble
Representation of StructureConformational Ensemble

Neither crystal nor solution structures can be properly represented by a single conformation

  • Intrinsic motions
  • Imperfect data

Variability reflected in the

RMSD of the ensemble

representation of structure

C

N

Representation of Structure

A representative conformer from the ensemble

variability uncertainty and flexibility in experimental structures

X-ray

NMR

  • Uncertainty

Ensemble 

Coord. Avg.

Avg. Coord.

+ B factor

  • Flexibility

Diffuse to 0 density

Mix static + dynamic

Less information

Sharp signals

Measure motions

Variability: Uncertainty and Flexibility in Experimental Structures
there is no such thing as a structure

Which is the biologically relevant conformer?

  • Does the molecule crystallize in the biologically relevant conformation?
  • What about proteins and protein machines who architecture is not fixed?
There is No Such Thing asA Structure!!!!
  • Polypeptides are dynamic and therefore occupy more than one conformation- structural dynamics
challenges for understanding the meaning of structure
Challenges For Understanding The Meaning of Structure
  • Structures determined by NMR, computation, and X-ray crystallography are static snapshots of highly dynamic molecular systems
  • Biological process (recognition, interaction, chemistry) require molecular motions (from femto-seconds to minutes)
  • New methods are needed to comprehend and facilitate thinking about the dynamic structure of molecules: visualize structural dynamics
visualization of structures
Visualization of Structures

Intestinal Ca2+-binding protein!

  • Need to incorporate 3D and motion
center for structural biology
Center for Structural Biology
  • Dedicated to furthering biomedical research and education involving 3D structures at or near atomic resolution
  • http://structbio.vanderbilt.edu