Biochemistry 300

Introduction to Structural Biology

Walter Chazin5140 BIOSCI/MRBIII

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

Jan. 7, 2009

• 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*

Organ Tissue Cell Molecule Atoms

Biology is Organized into Structures

MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIPREFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILIKENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASEMRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL

3D structure

What is Structural Biology?

Organism

CellSystem Dynamics

CellStructures

SSBs

polymerase

Assemblies

helicase

primase

Complexes

Sequence

Structural Scales

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

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

C BZn

A

NTD

14CTD

D

70NTD

70AB14/32D/70C

32CTD

Characterize Quaternary Structure

RPA70RPA70 RPA32RPA32RPA14RPA14

P

quaternary structure?

X-ray

NMR

Low-Res. Snapshots of Intact Protein(s)

MBP-tagged Siah-1

Inserting High Resolution Structures Into Low Resolution Envelopes

Mesh = DAMMINRibbon = 1QUQ

Techniques for Atomic Resolution Structural Biology

NMR Spectroscopy X-ray Crystallography

ComputationDetermine experimentally or model 3D structures of biomolecules

Structure Determined Differently by 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 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 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 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

NMR Spectroscopy X-ray Crystallography

ComputationDetermine 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

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

Representation of Structure

C

N

A representative conformer from the ensemble

Variability: Uncertainty and Flexibility in Experimental Structures

•Uncertainty

X-ray

Avg. Coord.+ B factor

NMR

Ensemble Coord. Avg.

•FlexibilityDiffuse to 0 densityMix static + dynamic

Less informationSharp signalsMeasure motions

There is No Such Thing asA Structure!!!!

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

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?

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

Center for Structural Biology

Dedicated to furthering biomedical

research and education involving 3D

structures at or near atomic resolution

http://structbio.vanderbilt.edu