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Biochemistry 301
Overview of Structural Biology Techniques
Jan. 19, 2004
MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIPREFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILIKENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASEMRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL
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