Biochemistry 301

Overview of Structural Biology Techniques

Jan. 19, 2004

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3D structure

Biological Structure

Organism

CellSystem Dynamics

CellStructures

SSBs

polymerase

Assemblies

helicase

primase

Complexes

Sequence

Structural Scales

• 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

High Resolution Structural Biology

High Resolution Structural Biology

Determine atomic structure

Analyze why molecules interact

Anti-tumor activityDuocarmycin SA

The Reward: UnderstandingControl

Shape

Atomic interactions

The Context of Atomic Structure

MoleculeStructural Genomics

PathwayStructural Proteomics

ActivitySystems Biology

RPARPA

NER

BER

RR

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

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*

Experimental Determination of 3D Structures

X-ray

X-raysDiffraction

Pattern

Direct detection ofatom positions

Crystals

NMR

RF

RFResonance

H0

Indirect detection ofH-H distances

In solution

Uncertainty and Flexibility inX-ray Crystallography and NMR

•Uncertainty

X-ray

Avg. Coord.+ B factor

NMR

Ensemble Coord. Avg.

•FlexibilityDiffuse to 0 densityMix static + dynamic

Less informationSharp signalsMeasure motions

Computational Problems3D Structure From Theory

• Molecular simulations

– Structure calculations (from experimental data)

– Simulations of active molecules

– Visualization of chemical properties to infer

biological function (e.g. surface properties)

• Prediction of protein structure (secondary

only, fold recognition, complete 3D)

Molecular Simulation

• Specify the forces that act on each atom

• Simulate these forces on a molecule and the responses to changes in the system

• Can use experimental data as a guide or an approximate experimental structure to start

• Many energy force fields in use: all require empirical treatment for biomacromolecules

Protein Structure Prediction:Why Attempt It?

• 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

Structure Prediction Methods

• Secondary structure (only sequence)• Homology modeling• Fold recognition• Ab-initio 3D prediction: “The Holy Grail”

1 QQYTA KIKGR

11 TFRNE KELRD

21 FIEKF KGR

Algorithm

Homology Modeling• Assumes similar (homologous) sequences

have very similar tertiary structures

• Basic structural framework is often the same (same secondary structure elements packed in the same way)

• Loop regions differ

–Wide differences, even among closely related proteins

Ab-Initio 3D Prediction• Use sequence and first principles of

protein chemistry to predict 3D structure

• Need method to “score” (energy function) protein conformations, then search for the conformation with the best score.

• Problems: scoring inexact, too many conformations to search

Complementarity of the 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- fundamental understanding of structure, dynamics and interactions (provides the why answers); models without experiment; very fast

Challenges for Interpreting3D Structures

• To correctly represent a structure (not a model), the uncertainty in each atomic coordinate must be shown

• Polypeptides are dynamic and therefore occupy more than one conformation–Which is the biologically relevant one?

Representation of Structure Conformational Ensemble

UncertaintyRMSD of the ensemble

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

Intrinsic motions

Imperfect data

Representations of 3D Structures

C

N

Precision is not Accuracy

Challenges for Converting3D Structure to Function

• 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: visualization*

Visualization of Structures

Intestinal Ca2+-binding protein!

Need to incorporate 3D and motion

Center for Structural BiologyThe Concept

Integrate the application of

X-ray crystallography, NMR, computational

and other complementary structural

approaches to biomedical problems

Center for Structural BiologyFacilities

• X-ray crystallography

Local facilities (generator + detectors)

Synchrotron crystallography

• NMR

Biomolecular NMR Center (2-500, 2-600, 800)

• Computation/Graphics

Throughput computing clusters

Resource Center Graphics Laboratory

Center for Structural BiologyA Resource

• Education and project origination

• Open-access (BIOSCI/MRBIII- 5th floor)

• Expertise (Laura Mizoue, Jarrod Smith +

Joel Harp- Xray & Jaison Jacob-NMR)

• Access to instrumentation to determine and

visualize structures

• Biophysical characterization- CD,

fluorescence, calorimetry