Refining the conformational ensembles of flexible proteins using - - PowerPoint PPT Presentation
Refining the conformational ensembles of flexible proteins using simulation-guided spectroscopy Jennifer M. Hays Blue Waters Symposium June 3 2018 Acknowledgements Columbus Lab Kasson Lab Linda Columbus, PhD Peter Kasson, MD,
Jennifer M. Hays Blue Waters Symposium June 3 2018
X-ray crystallography MD Simulations FRET DEER
Integrate experimental data using computation
I t
Perform experiments Identify informative experiments
FEMS Microbiol Rev. 2011;35(3):498-514
resonance (DEER) provides distance distributions between pairs of spin-labeled amino acids
heterogeneity of Opa ensemble
a few DOF in the system, but we need on the order of ~10,000 measurements
We want two things from
the conformational ensemble
with each other
Integrate experimental data using computation
I tPerform experiments Identify informative experiments
b)
(maximum-relevancy, minimum redundancy) Hays et al., Submitted
Ensemble distribution Experimental distribution
. . .
→→
→
→ →
→
→
Update the pull potential: Restart simulations
Integrate experimental data using computation
I tPerform experiments Identify informative experiments
b) a)
Distance (nm) Probability
MD Initial DEER 31 - 166 88 - 162 77 - 107 117 - 107
Hays et al., Submitted
After 100 ns of simulation per ensemble member (2 μs aggregate) , approximately converge to target
Distance (A) Probability
Time (ns) Time (ns)
A) B)
Probability
J-S Divergence J-S Divergence
DEER 2.5 ns 100 ns 200 ns
Integrate experimental data using computation
I tPerform experiments Identify informative experiments
Current method not robust enough to handle well-separated probability modes Come see my poster tonight for more details!
SSP mRMR: 1st round mRMR: 2nd round
40 30 20 10
33-159 39-176 90-172 38-163 36-91 85-171 23-163 82-166 Inverse J-S Divergence Hays et al., Submitted
C)
SV extended: 40 % HV2 extended: 20 % Multiple loop extensions: 40 %
1
Hays et al., Submitted Opa loops have three regions of high variability: SV (tan), HV1 (green) , HV2 (red)
A) B)
28 - 159 (SV - HV2) 80 - 166 (HV1 - HV2)
50.6 A 45.2 A
THR159 ASN166 ASN80 GLU28 Distance (A)
HV2 HV1 SV
Hays et al., Submitted
DEER pairs into MD simulation, we have obtained a conformational ensemble of apo Opa that suggests clear possible modes of CEACAM engagement
ensemble
which loop(s) CEACAM binds: HV2
DEER experiments Restrained- ensemble simulations Incorporate distributions into simulations Identify highly informative pairs
I t
4 2
a) b) c) d)
Pair-confjguration MI
200 200
a) b)
Opa 31-166 Opa 88-162 Opa 77-107 Opa 107-117 162 166 88 31 107 117 77
High conformational heterogeneity pairs Low conformational heterogeneity pairs
t (μs) Distance (nm) Intensity t (μs) Distance (nm) 2 entropy = 4.71 nats entropy = 4.64 nats entropy = 4.45 nats entropy = 4.34 nats
0.95 1.00
2
0.95 1.00 0.90 1.00
Intensity 2 4
1.00 0.90
4 2
DEER experiments Restrained- ensemble simulations Incorporate distributions into simulations Identify highly informative pairs
I t 20 30 10
mRMR: 2nd round mRMR: 1st round SSP Dimensionality of conformational ensemble
2 4 6
Information-theoretic coarseness (1-ε)
8 10
x10-5 Coarse Fine