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NESG NMR Conference Call Applications of RDCs and Use of Lanthanide Tags Presenter: J. Prestegard,...
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Transcript of NESG NMR Conference Call Applications of RDCs and Use of Lanthanide Tags Presenter: J. Prestegard,...
NESG NMR Conference Call
Applications of RDCs andUse of Lanthanide Tags
Presenter: J. Prestegard, UGA
9/26/05
Residual Dipolar Couplings in Structure Determination – Recent Reviews
• Prestegard, A-Hashimi & Tolman, Quart. Reviews Biophys. 33, 371-424 (2000).
• Bax, Kontaxis & Tjandra, Methods in Enzymology, 339, 127-174 (2001)
• Prestegard, Bougault & Kishore, Chemical Reviews, 104, 3519-3540 (2004)
• Lipsitz & Tjandra, Ann. Rev. Biophys. Biomol. Struct., 33, 387-413 (2004)
• Fushman et al., Prog. NMR Spect. 44, 189-214 (2004)
RDCs Come From the Dipolar Interaction Between Two Spins
15N
1HB0
r
HZNZ
2
3II
2
1θ3cos
r
C D
Brackets denote averaging – goes to zero without partial orientation
Inducing Order Using Liquid Crystalline MediaRestores Dipole Interaction in Solution
B0
Most versatile medium: C12E5:octanol
-1/4(J + D)
1/4(J + D)
-1/4(J + D)
1/4(J + D)E
J + D
RDCs are Easily Measured as Contributions to Multiplet Splittings
Some Other Experiments for 15N-1H RDC Measurement
• Tolman JR, Prestegard JH: Measurement of one-bond amide N-15-H-1 couplings. J. Magn. Reson., 1996, 112:245-252
• Ottiger M, Delaglio F, Bax A, Measurement of couplings using IPAP. JMR 1998,131: 373-378.
• Kontaxis G, Clore GM, Bax A, TROSY-HSQC offsets. J. Magn. Reson., 2000 143:184-190.
Use of 15N-1H RDCs for Structure Validation and Refinement: TM112
rmsd = 1.693(NMR vs Crystal structure)
REsidual Dipolar Coupling Analysis Tool(REDCAT)
Valafar, H., & J.H. Prestegard (2004), J. Mag. Res. 167: 228-241
• Given a proposed structure and RDCs, calculates order tensor solutions.
• Finds best order tensor solution.
• Gives principal elements and Euler angles.
• Back-calculates RDCs.
• Estimates errors and helps identify problematic data.
Another program: Dosset, Hus, Marion & Blackledge (2001), JBNMR, 20: 223-231
Correlation of Experimental 1H-15N RDCs with Calculated RDCs from the NMR Structure of TM112
NMR model after correction (Qfactor=0.57773)
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-8
-6
-4
-2
0
2
4
6
-30 -25 -20 -15 -10 -5 0 5 10
expt RDC
ca
lc R
DC
Correlation of Experimental 1H-15N RDCs with Calculated RDCs from Crystal Structure of TM1112
x-Ray model after correction (Q-factor=0.373893)
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-4
-2
0
2
4
6
-30 -20 -10 0 10
expt RDC
ca
lc R
DC
Structure Refinement Using RDCsSchwieters CD et al. XPLOR-NIH, J. Magn. Res. 160 (1): 65-73 JAN 2003
Write RDCs in principal alignment frame:D = (Da/r3){(3cos2θ – 1)/r3 + (3/2)Rsin2θcos(2)}
Write error function in terms of Dmeas and Dcalc
ERDC = (Dmeas – Dcalc)2
Seek minimum in ERDC to refine structure –Need to float alignment axes during search
Example of Validation and Refinement – MTH1743
plot of exp RDC vs calc RDC (Q-factor=0.519)
-10
-8
-6
-4
-2
0
2
4
6
8
10
-20 -15 -10 -5 0 5 10 15 20
exp RDC
ca
lc R
DC
expt RDC vs calc RDC after simulated annealing (2000K-20K) Q-factor = 0.157
-12
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-8
-6
-4
-2
0
2
4
6
8
10
-15 -10 -5 0 5 10
expt RDC
calc
RD
C
RMSD Before refinement : 0.711, After refinement: 0.672
Using RDCs directly in structure determination:PF1455 – A Protein with a Novel Fold?
• 10 kDa protein from Pyrococcus furiosus • No significant sequence identity to PDB
entries• No significant threading hits with
Genthreader or Prospect• RDC-Prospect finds a structural homolog• Can RDCs and backbone NOEs lead from
the homolog to a structure?
Data Collected:
• H-N RDCs – in phage - 63
• HaCa RDCs in phage - 61
• H-N RDCs – in C12E5 - 54
• Primary NOEs (45 sequential, 9 long range)
• Secondary NOEs (18 sequential, 19 long range)
• Ca shifts – 74
Refinement
• Started with 1cc8 as template• Constraints: NOE, RDC, torsion, radius of
gyration• Three rounds of simulated annealing (400K) in
vacuum • One round of simulated annealing in water• 20 independent runs – 1.4Å cluster• Structure moves 2.5Å rmsd from template• Ramachandran statistics: 56%, 30%, 13%, 1%
Plot of exp NH RDC vs calc RDC Q-factor = 0.14
-12
-10
-8
-6
-4
-2
0
2
4
6
8
-15 -10 -5 0 5 10
exp NH RDc
ca
lc N
H R
DC
T6_initial structure_ML51 (Q-factor = 0.75)
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-4
-2
0
2
4
6
-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8
exp NH RDC
calc
NH
RD
C
Comparison of RDC (phage HN) before and after refinementpink symbols excluded – validation set
Structure of PF1455
RDCs can be Collected Without Alignment Media: Lanthanide Tagged Proteins:
RDC = -(hB2)/(1203r3kT) [½Δ(3cos2θ-1) + ¾sin2θcos]
Note: B2 dependence
Ln3+
1 2
3
Ikegami, T., et al. (2004) J. Biomol. NMR 29:339-349.Wohnert, J., et al. (2003) J. Am. Chem. Soc. 125:13338-13339.
Construct for Lanthanide Tagged EB1
MGHHHHHHG*ENLYFQG**YIDTNNDGWYEGDELLA*SAVVYSTSVTSDNLSRHDMLAWINESLQLNLTKIEQLCSGAAYCQFMDMLFPGSIALKKVKFQAKLEHEYIQNFKILQAGFKRMGVDKIIPVDKLVKGKFQDNFEFVQWFKKFFDANYDGKDYDPVAARQGQETAVAPSLVAPALNKPKKPLTSSSAAPQRPISTQRTAAAPKAGPGVVRKNPGVGNGDDEAAELMQQVNVLKLTVEDLEKERDFYFGKLRNIELICQENEGENDPVLQRIVDILYATDEGFVIPDEGGPQEEQEEY
Wohnert, J., et al. (2003) J. Am. Chem. Soc. 125:13338-13339.
TROSY-HSQC correlations give RDC data.900 MHz Field-Induced Alignment
Paramagnetic Systems Give Other Complementary Information
Bertini, I., et al. (2002). Concepts in Magnetic Resonance 14: 259-286.
PCS 1
12r 3[ ax(3cos 2 1) rh sin2 cos2]
PRE 15
(o
4)2 1
r 6
Bo2H
2 (gJB )4 J 2(J 1)2
(2kBT )2(4 r 3 r
1 H2 r
2)
CCR 1
30(
o
4)2 Bo
2H2 (gJB )2 J(J 1)
rNH3 kBT
(3cos2 1)
r3(4 r 3 r
1 H2 r
2)
RDC 1
120 2
Bo2HS2
rNH3 kBT
[ax(3cos2 1) rh sin2 cos2]
Comparison of Lu3+ and Dy3+ Complexes of Tagged Q15691 gives Pseudo-Contact Shifts
CharacteristicDiagonal shifts
15-20 Å
20-25 Å
Ln3+
Paramagnetic Enhancement of Spin Relaxation:
Distance MappingOver 30Å
Provides Validation of Assignments
Lanthanide -Tagged Hum-Q-15691
Test Case:Assignment of Glycine Subset of
Amino Acids in Q15691
RDC(Hz) PCS(Hz) Assign r(exp) r(model) -0.9 -103 Gly 58 19 28 1.2 -152 Gly 98 16 16 2.3 1 Gly 103 14 16 -3.0 -44 Gly 116 36 37 7.1 -86 Gly 138 21 25
Acknowledgements
T. WeldeghiorghisSilvia MariJohn GlushkaHomay ValafarGreg Benison
Fang TianNitin JainKristen MayerSonal BansalPeter Leblond
NIGMS
http://secnmr.org
Example of Multiple Coupling Experiment: Soft HNCA – E.COSY
Weisemann, Ruterhans, Schwalbe, Schleucher Bermel, Griesinger, J. Biomol. NMR, 4, 231-240, 1994
Soft HNCA E-COSY Spectra of 15N-Labeled 13C Natural Abundance Rubredoxin
C chemical shift, Ci to C
i-1 connectivity, 3J-HNH coupling, C-H, HNH and H
i-1HN dipolar coupling
Inadvertent Mixing of α and β States of Hα can give Systematic Errors in J+D
+ f =
J+D too small J+D correct
Inclusion of RDCs Improves Accuracy of Structures
No Dipolar Data With Dipolar Data
Structure Precision (Å) Accuracy (Å) Precision (Å) Accuracy (Å)
GB1 2·88 4·33 1·37 1·12
BAF 1·31 1·37 1·23 0·91
CVN 1·67 1·53 1·23 1·10
FK506 0·67 1·12 0·29 0·74
GATA-1 0·76 NA 0·68 NA
KH 0·39 NA 0·16 NA
SAΔ41 0·71 NA 0·65 NA
3 1
23
2cos cos cos cos cos
i j k l kl
Order Matrix Analysis
x y
z
z
x
z
Finding a Principal Order Frame
Sxx Sxy ..Syx Syy .... .. ..
= ASx’x’
Sy’y’
Sz’z’
A-1
Simple Modification of Soft HNCA – E.COSYAllows scaled addition of sum and difference E.COSY
to compensate for relaxation effects
Weisemann, Ruterhans, Schwalbe, Schleucher Bermel, Griesinger, J. Biomol. NMR, 4, 231-240, 1994
+/- 90