Biochemistry 301

Jan. 12, 2003

Overview of Structural Biology Techniques

Sequence MESDAMESETMESSRSMYN AMEISWALTERYALLKINCAL LMEWALLYIPREFERDREVIL MYSELFIMACENTERDIRATV ANDYINTENNESSEEILIKENM RANDDYNAMICSRPADNAPRI MASERADCALCYCLINNDRKI NASEMRPCALTRACTINKAR KICIPCDPKIQDENVSDETAVS WILLWINITALL Organism

Biological Structure

3D structure Complexes Structural Scales helicase polymerase SSBs primase Assemblies Cell Structures System Dynamics Cell

High Resolution Structural Biology Organ

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Tissue

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Cell

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Molecule

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Atoms

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A cell is an organization of millions of molecules

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Proper communication between these molecules is essential to the normal functioning of the cell

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To understand communication: *

Determine the Arrangement of Atoms

*

High Resolution Structural Biology Determine atomic structure Analyze why molecules interact

The Reward: Understanding

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Control Anti-tumor activity Duocarmycin SA Atomic interactions Shape

How Atomic Structure Fits In RPA NER BER RR Molecule Structural Genomics Pathway Structural Proteomics Activity Systems Biology

The Strategy of Atomic Resolution Structural Biology

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Break down complexity so that the system can be understood at a fundamental level

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Build up a picture of the whole from the reconstruction of the high resolution pieces

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Understanding basic governing principles enables prediction, design, control

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Pharmaceuticals, biotechnology

Approaches to Atomic Resolution Structural Biology

NMR Spectroscopy X-ray Crystallography Computation

Determine 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 NMR X-rays Diffraction Pattern

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Direct detection of atom positions

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Crystals RF RF Resonance H 0

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Indirect detection of H-H distances

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In solution

Uncertainty and Flexibility in X-ray Crystallography and NMR X-ray NMR

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Uncertainty Avg. Coord.

+ B factor

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Flexibility Diffuse to 0 density Mix static + dynamic Ensemble

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Coord. Avg.

Less information Sharp signals Measure motions

Computational Problems

3D Structure From Theory

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Molecular simulations

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Structure calculations (from experimental data)

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Simulations of active molecules

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Visualization of chemical properties to infer biological function (e.g. surface properties)

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Prediction of protein structure (secondary only, fold recognition, complete 3D)

Molecular Simulation

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Specify the forces that act on each atom

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

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

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

Protein Structure Prediction:

Why Attempt It?

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A good guess is better than nothing!

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Enables the design of experiments

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Potential for high-throughput

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Crystallography and NMR don’t always work!

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Many important proteins do not crystallize

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Size limitations with NMR

Structure Prediction Methods

1 QQYTA KIKGR 11 TFRNE KELRD 21 FIEKF KGR Algorithm

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

Homology Modeling

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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

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Wide differences, even among closely related proteins

Ab-Initio 3D Prediction

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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.

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Problems: scoring inexact, too many conformations to search

Complementarity of the Methods

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

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NMR- enables widely varying solution conditions; characterization of motions and dynamic, weakly interacting systems

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

Challenges for Interpreting 3D Structures

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

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Polypeptides are dynamic and therefore occupy more than one conformation

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Which is the biologically relevant one?

Representation of Structure

Conformational Ensemble

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

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Intrinsic motions

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Imperfect data Uncertainty RMSD of the ensemble

Representations of 3D Structures C N Precision is not Accuracy

Challenges for Converting 3D Structure to Function

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Structures determined by NMR, computation, and X ray crystallography are static snapshots of highly dynamic molecular systems

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Biological process (recognition, interaction, chemistry) require molecular motions (from femto seconds to minutes)

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*New methods are needed to comprehend and facilitate thinking about the dynamic structure of molecules: visualization*

Visualization of Structures

Intestinal Ca 2+ -binding protein!

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Need to incorporate 3D and motion

Center for Structural Biology:

The Concept Completely integrate the application of X-ray crystallography, NMR and computational structural approaches to biological and biomedical problems

Center for Structural Biology

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X-ray crystallography Local facilities (generator + detectors) Synchrotron crystallography

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

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Computation/Graphics Throughput computing clusters Resource Center Graphics Laboratory

Structural Biology Resource (Not a Traditional Core!)

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Education and project origination

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

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Expertise (Laura Mizoue, Jarrod Smith, X)

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Hardware to determine and visualize structures (+ biophsysical characterization)