Biochemistry 300

Introduction to Structural Biology

Walter Chazin5140 BIOSCI/MRBIII

E-mail: Walter.Chazin@vanderbilt.eduhttp://structbio.vanderbilt.edu/chazin/classnotes/

Jan. 11, 2010

• 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 the basis for communication it is necessary to define the

atomic structures of the molecules and elucidate the fundamental forces driving interactions

Organ Tissue Cell Molecule Atoms

Biology is Organized into Structures

3D structure

What is Structural Biology?

Organism

Cell

CellStructures

SSBs

polymerase

Assemblies

helicase

primase

ComplexesAtoms

Multiple scales

- N- N - C- CO

HH

R

Determine atomic structure to analyze why molecules interact

Atomic Resolution Structural Biology

Anti-tumor activityDuocarmycin SA

The Reward: UnderstandingControl

Shape

Atomic interactions

Atomic Structure in Context

MoleculeStructural Genomics

PathwayStructural Proteomics

ActivityStruct. Systems Biol.

RPARPA

NER

BER

RR

Techniques for Atomic Resolution Structural Biology

NMR Spectroscopy X-ray Crystallography

ComputationDetermine experimentally or model 3D structures of biomolecules

Structure is Determined Differentlyby X-ray and NMR

X-ray

X-raysDiffraction

Pattern

Direct detection ofatom positions

Crystals

NMR

RF

RFResonance

H0

Indirect detection viaH-H distances

In solution

Why Structural Analysis 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 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

• Secondary structure (only sequence)• Homology modeling (using related

structure)• Fold recognition• Ab-initio 3D prediction: “The Holy Grail”

1 QQYTA KIKGR

11 TFRNE KELRD

21 FIEKF KGR

Algorithm

Computational ApproachesStructure Prediction

Complementarity of Methods

• X-ray crystallography- highest resolution structures; faster than NMR

• NMR- in solution; enables widely varying conditions; can characterize dynamic, weakly interacting systems and movement

• Computation- models without experiment; very fast; fundamental understanding of structure, dynamics and interactions; provides insight into driving forces

There is No Such Thing as A Structure!

• Polypeptides are dynamic and therefore occupy more than one conformation- structural dynamics

Is there a specific biologically relevant conformer?

Does a molecule crystallize in a biologically relevant

conformation?

What about proteins and protein machines which have

architecture that is not fixed?

Molecules are Dynamic, Not Static Conformational Ensemble

Variability reflected in the RMSD of the ensemble

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

Intrinsic motions

Imperfect data

Representing Molecular Structure

C

N

A representative conformer from the ensemble

How is Motion Reflected in X-ray Crystallography and NMR?

•Uncertainty

X-ray

Avg. Coord.+ B factor

NMR

Ensemble Coord. Avg.

•FlexibilityDiffuse to 0 densityMultiple occupancyMix static + dynamic

Sharp signalsFewer interactionsMeasure motion!

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

Intestinal Ca2+-binding protein!

Need to incorporate 3D and motion

The Divide and Conquer Strategy

• Cellular machinery has large and complicated structures not readily amenable to high resolution techniques

• Characterize the stable folded domains at the atomic level and elucidate driving forces

• Build up a structural model of the whole from a reconstruction with the high resolution pieces

Validate by experiments on the intact protein(s) and functional analysis

QuickTime™ and aAnimation decompressor

are needed to see this picture.

Protein Machines are DynamicProtein Machines are DynamicActivity Requires Remodeling of Multi-Protein AssembliesActivity Requires Remodeling of Multi-Protein Assemblies

C BZn

A

NTD

14CTD

D

70NTD

70AB14/32D/70C

32CTD

Protein Architecture

RPA70RPA70 RPA32RPA32RPA14RPA14

P

quaternary structure?

X-ray

NMR

Dynamic Architecture of Proteins in a Cell’s Molecular Machines

Movement/remodeling of architecture is intrinsic to function!!

Need Additional Techniques to Fill in the Gaps for Large Systems

NMR Spectroscopy X-ray Crystallography

ComputationDetermine experimentally or model 3D structures of biomolecules

• EPR/Fluorescence to measure distances when traditional methods fail

• EM and Scattering to get snapshots of whole molecular structures(Cryo-EM starts to approach atomic resolution!)

Snapshots of Molecular AssembliesVery large structures lower resolution

MBP-tagged Siah-1

Stewart Lab

Inserting High Resolution Structures into Low Resolution Envelopes

Mesh = DAMMINRibbon = 1QUQ

Center for Structural Biology

Dedicated to furthering biomedical

research and education involving 3D

structures at or near atomic resolution

http://structbio.vanderbilt.edu