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A N D R E W H E I N D E L
J A M E S M A D I S O N U N I V E R S I T Y
D R . J O S E P H C U R T I S
Computational Methods to Analyze Small Angle X-ray Scattering Data of Biological Molecule & Free Energy Calculations to
Validate Structural Ensembles
Ig58/59 Hfq + rpoS RNA
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Overview
• Background – Obscurin
• Advanced analysis • Ig58/59
• Free energy calculations• Ig58/59
• CHD1
• rpoS RNA/HFQ
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Muscle contraction
1
2
3
4
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Obscurin
• Cell maintenance + organization + signaling
• Ig58/59• Mutation (Arg8Gln)
Figure adapted from Kontrogianni-Konstantopoulos et al. (2009)Figure adapted from Fukuzawa et al. (2008)
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Intrinsic Flexibility/Disorder
Intrinsically disordered proteins Lack fixed 3-D structure
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Small Angle Scattering Theory
http://iramis.cea.fr/scm/lions/techniques/saxs/
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Modeling Methods in SAXS/SANS
Ensemble methods Approaches
Dummy atom
Atomistic modeling
http://en.wikipedia.org/wiki/Biological_small-angle_scattering
Dummy atom model
Atomistic model
Adapted from Clark et al. (2013)
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Data reduction of Ig58/59
Processed Scattering Data
Truncated Scattering Data
Potential non-ideality
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Guinier PlotTruncated Scattering
Data
Ig58/59 Analysis
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Structure Generation
Figures adapted from Computer Physics Communication 183 (2012) 382-389
Figure adapted from http://www.bmb.uga.edu/wampler/tutorial/prot2.html
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Ig58/59 Analysis
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Chi-squared Analysis
30.36Å
Ig58/59 Analysis
Rg (A) = 30.092 ± 4.373
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Ig58/59 Density Map
2-Dimensional Example
0
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Free Energy Calculations
Problem : Computationally intensive to calculate free-energy in MMC
Solution :
(ΔG) = molecular mechanical + solvation free energy
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Free Energy Calculations
Sample generated DCD trajectory
EM
MD production
MD equilibration
Solvate and ionize
Energy minimization (EM)
APBS
Extract DCD frames
1. Fixed protein atoms2. Fixed protein backbone3. Fixed secondary structure4. No constraints
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Conclusions
SAXS data suggests extended structure Contamination in sample
Intrinsic motion within globular domains
Future directions SANS (8/21)
Free energy calculations Study ongoing
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Acknowledgements
Dr. Joseph Curtis
Dr. Julie Borchers
Dr. Susan Krueger
Dr. Nicholas Clark
Dr. Nathan Wright
James Madison University
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Free Energy Systems
Ig58/59
Chd1 (Wt) + Chd1 (mutant) ATP-dependent chromatin remodelers
Disrupt DNA binding
Hfq + rpoS RNA leader Accelerates base pairing between sRNA &
mRNA
Provides translational control
Hfq + rpoS RNA
Chd1 (wt) Chd1 (mut)
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Overview
• Small angle X-ray scattering (SAXS) data sets • Ubiquitin-Aar
• Ig1/2
• Ig58/59
• SAXS analysis • Ig58/59
• Free energy calculations• Ig58/59
• CHD1
• rpoS RNA/HFQ
Ig1/2
Ub-Aar
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Ig58/59 Structure Generation
• Crystal structure
• NAMD vacuum minimization
• VMD solvation box
• VMD ionization
• NAMD minimization
• Fixed protein atoms
• Fixed protein backbone
• Fixed secondary structure
• No constraints
• NAMD/CHARMM molecular dynamics