Integrated Analysis of Molecular Assemblies using Chimera
A Demonstration with Alpha Crystallin
NCRR Site Visit
November 17, 2011
Purpose of this Demonstration
- This demonstration will show the inter-play of many current
Chimera capabilities to model a molecular assembly.
- It illustrates the interactive, integrated and multi-faceted nature
of the Chimera software package.
- Will show tools developed as part of the technology project called
"Software for interactive analysis of large molecular assemblies".
Software for Analysis of Large Molecular Assemblies
- Examples of large molecular assemblies are viruses, ribosomes,
polymerases, spliceosome, nuclear pores, proteasome, microtubules,
nucleosomes, and a few thousand more.
- Most proteins and nucleic acids in the cell carry out their functions
in dynamic large assemblies, often composed of tens of molecules, sometimes
hundreds of molecules.
- Chimera is the leading software for interactive model building
and analysis of large molecular assemblies.
- Chimera is used by nearly every research lab that studies assemblies
by 3-dimensional electron microscopy.
- Software development is driven by interactions with over
100 electron microscopy research labs.
Example System: Alpha Crystallin
- Human alpha crystallin is a molecular chaperone that prevents
aggregation of unfolded proteins.
- Its oligomeric structure and mechanism of functioning are poorly understood.
- About 50% of the protein in the eye lens is alpha crystallin.
- Alpha crystallin is found in many tissues, especially the heart and
brain, and is an important factor in diseases ranging from cataracts and
heart disease to neurodegenerative diseases where protein aggregation
occurs such as Parkinson's and Alzheimer's.
Modeling Alpha Crystallin
- This demonstration will build a model of an alpha crystallin 24-mer,
explore its mechanism of preventing aggregation, and compare it to a
distantly related Archaeal small heat shock protein 24-mer.
- Main message of the demonstration: a diverse collection of
software analysis capabilities is required for the analysis. The many
Chimera capabilities enable incorporating all available data in the
analysis (we will show EM, X-ray, NMR and SAXS).
- The entire model building takes 60 minutes of interactive work.
We will look at some of the steps in the 10 minutes available.
|24 copies of alpha crystallin form oligomer|
seen by electron microscopy (20 Angstrom resolution).
|Human alpha crystallin is 2 beta sheets.
How Alpha Crystallin Binds to Itself: Evidence from X-ray, NMR, SAXS
|Various crystal structures show
several biological dimerization modes.
||C-terminal tail binds to edge of beta-sheet.
Binds in 180 degree flipped orientation too.
Binding sequence is palindrome.
||Side-by-side beta sheet dimer.
Building the Model: Fitting and Optimization
|Deduce map symmetry.
||Fit dimer in EM map. Movie.
||Symmetry copies of fit dimer.
|Hexamer with 6 C-terminal tails.
||Moved C-terminal tails to bind hexamer.
||Made C-terminal tails connect 4 hexamers.
||Fit of 24-mer model to map.
Mechanism of Chaperone Activity: A Hypothesis
|Molecular surface with orange hydrophobic regions.
||C-terminal strands cover hydrophobic grooves.
Literature says binding unfolded proteins requires
oligomer to disassemble.
Comparison to an Archaeal Chaperone
|M jannaschii octahedral small heat shock protein 24-mer (1shs),
20% sequence identity to human alpha-crystallin.
||Same C-terminal tail binding pattern as our alpha-crystallin model.
||Alpha-crystallin is larger. Two inequivalent of 3-fold axes
in alpha-crystallin are equivalent in M jannaschii sHSP.
|Aligned alpha-crystallin (red) and sHSP (blue) monomers.
||Aligned alpha-crystallin dimer (red), sHSP dimer (blue).
All 12 alpha-crystallin structures show edge-to-edge dimers. None
show sHSP type strand swapping.
||Comparison of alpha-crystallin EM map and simulated
map from sHSP crystal structure.
Chimera Capabilities used for this Example Analysis
We used 16 Chimera capabilities in this example, only about 1/5 of the
available Chimera molecular assembly modeling tools.
- Density map contour surface calculation and display.
- Enclosed volume measurement. Used to compute contour level to enclose expected volume of 24-mer.
- Crystal unit cell display.
- Connected sequence and structure views.
- Sequence based structure alignment.
- Small-angle x-ray scattering profile calculation.
- Fitting atomic models in density maps, global, symmetric.
- Map symmetry calculation.
- Symmetric replication of atomic molecules.
- Moving molecular fragments (C-terminal tails) with energy minimization.
- Solvent excluded surface calculation.
- Hydrophobicity surface color mapping.
- Density map simulated from atomic model (M jannaschii structure).
- Morphing between atomic model conformations.
- Fitting one density map into another.
- Morphing between density maps.
Main message of the demonstration
- Analyzing molecular assemblies proceeds by combining many Chimera capabilities.
- These enable factoring in all available experimental data:
electron microscopy, X-ray, NMR and SAXS.