Likai Song, Ilker Sen, Marco Bonora, - Cornell University
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Florida State University, NHMFL Likai Song, Ilker Sen, Marco Bonora, P. Fajer Ifs and Buts of DEER
Transcript of Likai Song, Ilker Sen, Marco Bonora, - Cornell University
Florida State University, NHMFL
Likai Song, Ilker Sen, Marco Bonora, P. Fajer
Ifs and Buts of DEER
11/06/09
1. X-band v. W-band: is bigger better ?• DEER• STEPR
2. DEER Analysis: how not to over interpret the data ?
3. Rotamers: is there H-bond ?
4. Structure determination: EPR as a structural method ?
0.17
0.18
0.17
0.26
0.24
0.027
0.026
0.010
0.064
0.058
X-band W-band
CDB3_341C
CDB3_342C
CDB3_142C
T4L_21C65C
TnC_55C83C
W-band DEER
High frequency (W-band) DEER
0 1 2
0 1 2
2 3 4 5 6 7
2 3 4 5 6 7
us
us
nm
nm
9.5 GHz
95 GHz
DEER spectra Distance distribution15
25
35
45
15 25 35 45
Distance X-band (A)D
ista
nce
W-b
and
(A)
W-band v. X-band distances
Likai Song, J. Zhou & P. Fajer‘09
Excellent agreement between the X- and W-band determined distances on three different proteins.
Band 3
T4 LysozymeTroponin C
Presenter
Presentation Notes
High field DEER at 95 GHz offers a different Larmor frequency to sample the dipolar interactions. DEER at 9.5 GHz and at 95 GHz is shown on the left (in blue) The Gaussian modeled distance distributions are very similar at both frequencies (orange curves) Comparison of 6 double mutants of 3 different proteins (TroponinC, Band3 and T4 lysozyme) confirms 1:1 correlation between the X- and W-band distances. Thus one can use the two frequencies and analyze in “global” manner. Below is for you info – things that were advertised but we could not verify: Better sensitivity of W-band – in fact it is much worse (factor of 2) due to instrumental instabilitites and smaller fraction of excited spins (due to larger dispersion). (there is better senesitivity to a absolute number of spins so that we can have 100x less sample but who is nowadays limited by the sample. We are talking 10 nanomoles at X-band v 0.3 nanomolesat W-band. sensitivity to longer distances: there was hope that at W-band the spin coherence persists longer allowing longer mixing times and thus pulling out weaker dipolar interactions. Not so. Checked on 3 samples. Phase cohrence was identical. Orientational selectivity: larger spectral dispersion translates into ability to pump selected orientation and observe another orientation. We are working on it but preliminary data would suggest that the lower sensitivity at W-band will make the orientational selection experiments v. difficult if possible.
A B
Dipolar frequency Dipolar frequency
Orientational selectivity22o disorder
Denysenkov, Prisner, Bennati, PNAS ’06Savitsky, Dubinskii, Flores, Lubitz, and K. Mbius, JPC ’07Polyhach, Godt, Bauer G. Jeschke, JMR ‘07
T4L_65C89C
Time (ns)
- 30 G (0.012)
- 20 G (0.016)
- 10 G (0.037)
Time (ns)
+ 35 G (0.021)
+ 30 G (0.026)
+ 25 G (0.023)
+ 15 G (0.048)
+ 10 G (0.062)
Pump-observe field scan
No orientational selectivity observed
ST-EPR at W-bandSpectral diffusion sensitivity
Spectral diffusion is 4-fold greater at W-band than at X-band
W-band ST-EPR is 4 times more sensitive to slow motion (us–ms)
DEER analysis – DeFit
Sen & Fajer EPR newsletter ’09
Excellent definition of complex distribution
F-test decision on # gaussians
Multiple populations
Add noise
Add error contours !
2
1
34
Wid
th (n
m)
2
1
34
Wid
th (n
m)
Distance (nm)
2 4 6
When two populations are different ?
Spin label rotamer simulation
No modelling
0
10
20
30
40
50
0 10 20 30 40 50Cβ-Cβ (A)
EPR
spi
n-sp
in (A
)
CW KCsADEER apoTnC DEER Ca TnC DEER troponin Ca T1 apoTnC
Multiple spin-spin distances observed for a single x-ray distance
Modelling
y = 0.93x + 3.18R2 = 0.93
0
10
20
30
40
50
0 10 20 30 40 50
X-ray plus MD/MC (A)
EPR
spi
n-sp
in (A
)
CW KCsADEER apoTnC DEER Ca TnC DEER troponin Ca T1 apoTnC T1 troponin Ca
Adding modeling to x-ray structures now shows correlation between EPR and atomistic detail
Sale et al. JACS 2005, Fajer J. Phys. ‘07
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Simulated Scaling MethodHongzhi Li, Mikolai Fajer, and Wei Yang, JCP ‘06
• Potential energy is separated into a scaling portion (Us) and an environment portion (Ue)
• Scaling factor λm changes from 0 to 1 during simulation facilitating transitions
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Potential of Mean Force and X-ray
Spin labels buried in protein interiorVisualization
• SS correctly predicts the X‐tal conformation of a buried residue.
M. Fajer, JACS ‘07
Distribution: model v. data
observed distribution due to SL rotamer distribution
0
0.2
0.4
0.6
0.8
1
1 2 3 4 5 6
MC/MDDEER
model to get insight what is rotamer and what is backboneheterogeneity
MC/MD
observed distribution due to backbone distribution
DEER
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Hydrogen bonding in MTSSL ?
?
Mchaourab & Hubbell ’96, Columbus & Hubbell ‘02 : comparison of S-S and C-S linkages
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χ1-χ2 scan energy profile (χ3=90)
basis set independent (PM3, PM3MM, HF 6-31*)
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Molecular orbitals
NBO deletion (HF, DFT):the global minimum rotamer has ~1 kcal/mol stabilization energy
Lone pair of the Sδinteracts with H of Cα
11/06/09
QM rotamers and x-rayGuo, Hubbell Structure ‘08
Rotamer Energy S2-CaH S1-NH X-ray
mm 0 0.7 0.9 yes
mt 0.4 0.3 rare
tp 1.2 0.7 yes
tm 2.0 yes
11/06/09
Other secondary structures
Rotamer Energy S2-CaH S1-NH
tp 0 0.8
mm 1.2 0.5
tt 1.2
tm 1.4
Rotamer Energy S2-CaH S1-NH
mm 0 0.7 0.1
tp 0.8 0.5
tt 1.4
mp 1.3
mt 1.8
pt 1.7
Beta strand Loop
From EPR to structure
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DGEOM-lite - Monte Carlo/Simplex strategy
random starting position
evaluation of distances with EPR penalty function
best agreement with experimental restraints
Monte Carlo iterations
simplex
Scoring function is EPR distance
distribution
0 10 20 30 40 50 60 70
Interspin Distance (Å)
Penalty
1.0
0.8
0.6
0.4
0.2
0
-0.2
Eulerian rotations + translation
- manipulate few points (EPR spin labels)- use of DEER distance distributions
Structure from EPR
11/06/09 DUAL Simplex 1K weighted rdn2 30
6 restraints
Presenter
Presentation Notes
rotation
11/06/09 DUAL Simplex 1K weighted rdn2 31
rotation
Presenter
Presentation Notes
rotation
11/06/09 DUAL Simplex 1K weighted rdn2 32
rotation
Presenter
Presentation Notes
rotation
11/06/09 DUAL Simplex 1K weighted rdn2 33
rotation
Presenter
Presentation Notes
rotation
11/06/09 DUAL Simplex 1K weighted rdn2 34
translation
Presenter
Presentation Notes
translated balls
Smooth muscle myosin
Position of the solutions with χ2min < χ2 < 5 χ2
min
Best solution
χ2min = 1.2
our conformationEM
DGEOM-lite reproduces the EM structure
monomers
Wendt et al. model∗
χ2<1.5 <1.4 <1.3 <1.25 χ2min
monom
ers
best fit
∗ ∗
∗
Best 20 solutions
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Collaborators
Likai Song (Harvard)
Ilker Sen (A. Einstein U.)
Wei Yang (FSU)
Mikolai Fajer (now at UCSD)
