Transcript BCHM 300 Introduction to Structural Biology (2011) lecture 1
Biochemistry 300
Jan. 5, 2011
Introduction to Structural Biology
Walter Chazin 5140 BIOSCI/MRBIII E-mail: [email protected]
http://structbio.vanderbilt.edu/chazin/classnotes/
Structural Biology- Multiple Scales
3D structure R - N - C
a
- CO H Atoms Organism Complexes helicase polymerase SSBs primase Assemblies Cell Structures Cell
The Underlying Basis for Biology
Organ
Tissue
Cell
Molecule
Atoms
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A cell is an organization of millions of molecules
•
Proper communication between molecules is essential to normal functioning of cells and miscommunication is the basis for disease
•
To understand the basis for communication it is necessary to define the atomic structures of the molecules and to elucidate the fundamental forces driving interactions between them
Atomic Resolution Structural Biology
Determine atomic structure to analyze why molecules interact
The Reward: Understanding
Control
Anti-tumor activity Duocarmycin SA Atomic interactions Shape
Atomic Structure in Context
RPA NER BER RR Molecule Structural Genomics Pathway Structural Proteomics Activity Struct. Systems Biol.
See commentary by SC Harrison, NSMB 11, 12-15 (2004)
Techniques for Atomic Resolution Structural Biology
NMR Spectroscopy X-ray Crystallography Computation
Determine experimentally or model 3D structures of biomolecules
Structures from X-ray Crystallography and NMR are Generated Differently X-ray NMR X-rays Diffraction Pattern
Direct detection of atom positions
Crystals RF RF Resonance H 0
Indirect detection via H-H distances
In solution
Why Compute Structures?
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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
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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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Invaluable for analyzing/understanding structure
Computational Approaches
Molecular Simulations
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Convert experimental data into structures
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Predict effects of mutations, changes in environment
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Insight into molecular motions
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Interpret structures- characterize the chemical properties (e.g. surface) to infer function
Computational Approaches
Structure Prediction
1 QQYTA KIKGR 11 TFRNE KELRD 21 FIEKF KGR Algorithm
• • • •
Secondary structure (only sequence) Homology modeling (using related structure) Fold recognition Ab initio 3D prediction: “The Holy Grail”
Complementarity of Methods
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X-ray crystallography- highest resolution structures; faster than NMR
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NMR- in solution; enables widely varying conditions; can characterize dynamic, weakly interacting systems and movement
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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 with architecture that is not fixed?
Molecules are Dynamic, Not Static
Conformational Ensemble “Neither crystal nor solution structures can be properly represented by a single conformation”
Intrinsic motions
Imperfect data Variability reflected in the RMSD of the ensemble
Representing Molecular Structure
C N A representative conformer from the ensemble
How is Motion Reflected 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 Multiple occupancy Mix static + dynamic Ensemble
Coord. Avg.
Sharp signals Fewer interactions Measure motion!
Challenges For Understanding The Meaning of Structure
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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: visualize structural dynamics
Visualization of Structures
Intestinal Ca 2+ -binding protein!
Need to incorporate 3D and motion
Addressing Complex Systems:
The Divide and Conquer Strategy
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Cellular machinery has large and complicated structures not readily amenable to high resolution techniques
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Characterize the stable folded domains at the atomic level and elucidate driving forces
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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
Need Additional Techniques For Large Molecules/Complexes
NMR Spectroscopy X-ray Crystallography Computation
Determine experimentally or model 3D structures of biomolecules
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EPR/Fluorescence to measure distances when traditional methods fail
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EM and Scattering to get snapshots of whole molecular structures (Cryo-EM starts to approach atomic resolution!)
Snapshots of Molecular Assemblies
Very large structures
lower resolution
MBP-tagged Siah-1
Stewart Lab
Inserting High Resolution Structures into Low Resolution Envelopes Mesh = DAMMIN Ribbon = 1QUQ
The Horizon: Dynamic Protein Machinery
Activity Requires Remodeling of Multi-Protein Assemblies
Thinking in Terms of Protein Architecture 14/32D/70C 70AB X-ray 32CTD P D 14
Zn
C B A CTD RPA70 RPA32 RPA14 NTD
quaternary structure?
70NTD NMR
Dynamic Architecture of Proteins in Molecular Machines
Movement/remodeling of architecture is intrinsic to function!!
Center for Structural Biology
Dedicated to furthering biomedical research and education involving 3D structures at or near atomic resolution http://structbio.vanderbilt.edu