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Jan. 11, 2010. Biochemistry 300 Introduction to Structural Biology. Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@vanderbilt.edu http://structbio.vanderbilt.edu/chazin/classnotes/. Biology is Organized into Structures. Organ Tissue Cell Molecule Atoms. - PowerPoint PPT Presentation
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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
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