NMR Facility Operations at NYSBC

NMR Facility
Operations at
NYSBC
© NYSBC 7 Sep 2006
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NYSBC
Introduction
The New York Structural Biology
Center is an independent
corporation acting as a resource
facility for its ten Member
Institutions.
ƒAlbert Einstein College of Medicine of Yeshiva University
ƒCity University of New York
ƒColumbia University
ƒMemorial Sloan Kettering Cancer Center
ƒMount Sinai School of Medicine
ƒNew York University
ƒRockefeller University
ƒState University of New York
ƒWadsworth Center, NYS Dept. Health/HRI
ƒWeill Cornell Medical College of Cornell University
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NYSBC operates cryo-electron microscopy
resources, two beam lines at NSLS/BNL, and a
large NMR facility. There are investigatorheaded laboratories, including the protein
preparation lab for the New York Consortium for
Membrane Protein Structure, funded by
NIGMS-PSI. The resources are predominantly
available to the Member Institutions, including
investigators:
Aneel Aggarwal
Mark E. Girvin
Carlos A. Meriles
Ruth Stark
David Allis
Paul Gottlieb
Gaetano Montelione
Thomas Szyperski
Clay Bracken
Steve Greenbaum
Tom Muir
Maria Luisa Tasayco
Esther Breslow
Clare Grey
Fred Naider
Peter Tonge
David Cowburn
Swapna V. Gurla
Arthur G. Palmer
Iban Ubarretxena-Belandia
Samuel J. Danishefsky
Griselda Hernandez
Dinshaw Patel
Chunyu Wang
Seth Darst
Barry H. Honig
Brian Phillips
Milton H. Werner
David Eliezer
Alexej Jerschow
Daniel Raleigh
Stanislaus Wong
John Spencer Evans
Tarun Kapoor
Thomas P. Sakmar
Lei Zeng
Jack H. Freed
David LeMaster
Nicole Sampson
Ming-Ming Zhou
Nicholas Geacintov
Chin Lin
Alexander Shekhtman
Martine Ziliox
Ranajeet Ghose
Min Lu
Samuel Singer
Lane Gilchrist
Ann McDermot
Steven Smith
Investigators listed are both direct users of the Facility and those collaborating with direct users.
Users from outside the NYSBC member institutions are co-PIs or users of the 900 MHz
Structural Biology Resource funded by NIH GM-66354
© NYSBC 7 Sep 2006
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NMR Resources
Approximately 35 research groups with
about 90 operators use the instruments
shown in tables below, plus 700, 600 &
500 systems.
Spectrometer
Probe
0.1%
Ethylbenzene
S/N
2mM Sucrose
1
Anomeric H
S/N
900 #1
cp TCI
8707:1
1006:1
US2
TCI
2440:1
495:1
800 US2 #1
cp TCI
7450:1
915:1
800 US2 #2
cp TCI
9011:1
851:1
800 conv
cp TXI
7050:1
940:1
900
#2
Probes
750 MHz
Tuning
Range, MHz
HXY
wide bore
X: 150-200
Y:70-130
HFX
wide bore
X:35-225
HCN
standard
bore
narrowband
HRMAS
HCND
narrowband
Fields, kHz.
1
H/13C/15N
90% B1
Homogeneity
Volume
LowerTemp.
Range, oC(3)
10kHz/15kHz
120/50/50
35-40 μl
-40/-30
120/50/50
35-40 μl
-40/-30
C 180:1
N: 30:1
120/50/50
35-40 μl
-15/-5
H : 250:1
90o pulse:
6μs/8μs/15μs
35-40 μl
n.a.
S/N(1,2)
13
C 160:1
N: 30:1
15
13
C 240:1
N: 30:1
15
13
15
1
(1) CPMAS probes: sensitivity is measured on a fully packed natural abundance glycine
sample, 4 scans, 1H-13C cross polarization experiment.
(2) HRMAS probes: sensitivity is measured on 60 μ volume sample of 0.1% ethylbenzene
solution in CDCl3.
(3) These probes have reliably achieved the sample temperatures at the indicated spinning
speeds using either an Airjet cooler or Bruker Cooling unit as the source of cold gas.
© NYSBC 7 Sep 2006
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Spectrometer Performance
Heteronuclear spin-echo difference test is run on a sample
containing 500 mM Sucrose in 100% D2O. In this test the
12C center-band of the sucrose anomeric protons is
suppressed in two scans, below the intensity of natural
abundance 13C side-bands in a series of 20 consecutive
experiments.
The lineshape stability test is run on a non-spinning
sample of 0.3% CHCl3 in Acetone-D6. The lineshape is
described by the hump numbers (linewidth at 0.55%/0.11%
of the carbon satellite peaks and half height of the main
chloroform peak). In this test the hump numbers should
not degrade by more than 10% over a period of 12 Hrs.
Protein Solid State Performance
Characteristic solid state spectra (left) on labeled ubiqutin. left – CP 13C/ 1H; center, double CP 13C/15N/1H; right, 2-D double CP
13C/15N/1H
© NYSBC 7 Sep 2006
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NMR Environment
Constructing a Center in Manhattan
with minimal interference from the
outside environment presents
challenges.
Red surface are at the 5 G contour
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To achieve excellent
vibration isolation, we
choose to take
advantage of Manhattan
geology, and provide
linkage of all critical
NMR and CEM sites to
the local hard schist
bedrock. For the 800’s
and 750 on a
suspended floor, this
involved constructing
four c. 3 m diameter
columns sunk to
bedrock, up to 15 m
high (left – picture of
one column).
© NYSBC 7 Sep 2006
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© NYSBC 7 Sep 2006
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NMR Results
Publications. (2005-6) see http://www.nysbc.org/papers
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Poget SF, Krueger-Koplin ST, Krueger-Koplin RD, Cahill SM, Chandra Shekar S, Girvin ME 'NMR Assignment of the Dimeric S. aureus Small
Multidrug-Resistance Pump in LPPG Micelles.' J Biomol NMR 2006 Feb 2
Shekar, S.C., Wu, H., Fu, Z., Yip, S.C., Cahill, S.M., Girvin, M.E., Backer, J.M. (2005) Mechanism of constitutive PI 3-kinase activation by oncogenic
mutants of the p85 regulatory subunit. J Biol Chem 280, 27850-27855
Yu, G. Vengadesan, H., Wang, L., Jashi, T. Yefremov, S. Tian, V. Gaba, I. Shomer and R. E. Stark, “Magic-Angle Spinning NMR Studies of Cell WallBound Aromatic-Aliphatic Biopolyesters Associated with Strengthening of Intercellular Adhesion in Potato ( Solanum Tuberosum L.) Tuber
Parenchyma,” Biomacromolecules 7, 937-944. Pujato M, Navarro A, Versace R, Mancusso R, Ghose R, Tasayco ML 'The pH-dependence of amide
chemical shift of Asp/Glu reflects its pK(a) in intrinsically disordered proteins with only local interactions.' Biochim Biophys Acta 2006 May 13
Meriles, CA, Dong W. "Indirect detection of nuclear magnetic resonance via geometrically induced long-range dipolar fields" J. Magn. Res. In Press,
Corrected Proof
Marulanda, D, Tasayco, M.L., Cataldi, M., Arriaran, V., Polenova, P. (2005) Resonance Assignments and Secondary Structure Analysis of E. coli
Thioredoxin by Magic Angle Spinning Solid-State NMR Spectroscopy. Journal of Physical Chemistry B. 109, 18135-18145.
Pujato, M.; Bracken, C.; Mancusso, R.; Cataldi, M.; Tasayco, M. L. pH-Dependence of Amide Chemical Shifts in Natively Disordered Polypeptides
Detects Medium-Range Interactions with Ionizable Residues. Biophysical Journal 89, 3293-3302.
Edwards , T.A., Butterwick, J.A., Zeng, L., Gupta, Y.K., Wang, X, Wharton, R.P., Palmer, A.G., Aggarwal, A.K. Solution Structure of the Vts1 SAM
Domain in the Presence of RNA J. Mol. Biol. 356, 1065-1072.
Tang Y, Grey MJ, McKnight J, Palmer AG, Raleigh DP 'Multistate Folding of the Villin Headpiece Domain.' J Mol Biol 355, 1066-1072.
Grey, M.J., Tang, Y, Alexov, E, McKnight, C.J., Raleigh, D.P.,Palmer, A.G. (2006) Characterizing a Partially Folded Intermediate of the Villin
Headpiece Domain Under Non-denaturing Conditions: Contribution of His41 to the pH-dependent Stability of the N-terminal Subdomain . Journal of
Molecular Biology 355, 1078-1094.
Valentine, E. R., and Palmer, A. G., 3rd (2005). Microsecond-to-Millisecond Conformational Dynamics Demarcate the GluR2 Glutamate Receptor
Bound to Agonists Glutamate, Quisqualate, and AMPA. Biochemistry 44, 3410-3417
Zech, S.J., Wand, A.J., McDermott, A.E. Protein Structure Determination by high resolution solid-state NMR spectroscopy: Application to
microcrystalline ubiquitin. J Am Chem Soc 127, 8618-8626.
Li H, Ilin S, Wang W, Duncan EM, Wysocka J, Allis CD, Patel DJ 'Molecular basis for site-specific read-out of histon H3K4me3 by the BPTF PHD finger
of NURF' Nature 442, 31-32
Teplova M, Yuan YR, Phan AT, Malinina L, Teplov A, Patel DJ 'Structural Basis for recognition and sequestration of UUU(OH) 3' termini of nascent
RNA polymerase II transcripts by LA, a rheumatic diseaase autoanitgen' Mol. Cell 21 75-85
Phan A.T., Kuryavyi V., Gaw H.Y., Patel D.J. (2005). Small-molecule interaction with a five-guanine-tract G-quadruplex structure from the human MYC
promoter. Nature Chemical Biology, 1, 167-173.
Zhang N., Phan A.T., Patel D.J. (2005). (3+1) assembly of three human telomeric repeats into an asymmetric dimeric G-quadruplex. J. Am. Chem.
Soc., 127, 17277-17285.
Zhang, N., Lin, C., Huang, X., Kolbanovskiy, A., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (2005). Methylation of Cytosine
at C5 in a CpG Sequence Context Causes a Conformational Switch of a Benzo[a]pyrene diol epoxide-N(2)-guanine Adduct in DNA from a Minor
Groove Alignment to Intercalation with Base Displacement. J Mol Biol 346, 951-965.
Phan A.T., Kuryavyi V., Ma J.B., Faure A., Andréola M.L., Patel D.J. (2005). An interlocked dimeric parallel-stranded DNA quadruplex: A potent
inhibitor of HIV-1 integrase. Proc. Natl. Acad. Sci. USA, 102, 634-639.
Phan A.T., Modi Y.S., Patel D.J. (2004). Propeller-type parallel-stranded G-quadruplexes in the human c-myc promoter. J. Am. Chem. Soc. , 126,
8710-8716.
Serganov A., Yuan Y.R., Pikovskaya O., Polonskaia A., Malinina L., Phan A.T., Hobartner C., Micura R., Breaker R.R., Patel D.J. (2004) Structural
basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. , 11, 1729-1741.
Qian C, Zhang Q, Li S, Zeng L, Walsh MJ, Zhou MM. (2005)Structure and chromosomal DNA binding of the SWIRM domain. Nat Struct Mol Biol. 2005
12 (12):1078-85. Edwards, TA, Butterwick, JA, Zeng, Z, Gupta, YK, Wang, X, Wharton, RP, Palmer, AG, Aggarwal, AK. Solution Structure of the Vts1
SAM Domain in the Presence of RNA J. Mol. Biol. 365, 1065-1072.
Carrington PE, Sandu C, Wei Y, Hill JM, Morisawa G, Huang T, Gavathiotis W, Yu W, Werner MH "The structure of FADD and its mode of interaction
with procaspase-8" Mol. Cell 22 599-610
Ji, H., Shekthman, A., McDonnell, J., Ghose, R., Cowburn D. (2006) "NMR determination that an extended BH3 motif of pro-apoptotic BID is
specifically bound to BCL-XL " Magnetic Res. Chem. 44 101-6
Muralidharan V., Dutta K., Cho J., Vila-Perello M., Raleigh DP., Cowburn D., Muir TW 'Solution Structure and Folding Characteristics of the C-Terminal
SH3 Domain of c-Crk-II' Biochemistry 45, 8874-8884.
Burz, D.S., Dutta, K., Cowburn, D., Shekhtman, A. (2006) Mapping structural interactions in proteins using NMR (STINT-NMR). Nature Methods 3, 9395; Nature Protocols, in press.
Tang Y, Goger MJ, Raleigh DP 'NMR Characterization of a Peptide Model Provides Evidence for Significant Structure in the Unfolded State of the Villin
Headpiece Helical Subdomain.' Biochemistry 2006 Jun 6;45(22):6940-6946
Peng, L., Liu, Y., Kim, N., Readman, J.E., and Grey, C.P. Detection of Brønsted acid sites in zeolite HY with high-field 17O-MAS-NMR techniques.
Nature Materials 4 216-219
Deng, Y, Liu, J, Zheng, Q, Eliezer, D, Kallenbach, N, Lu, M. Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hyrdrophobic Heptad Repeat.
Structure 14 (2006), 247-255.
Deng, Y, Liu, J, Zheng, Q, Yong, W, Lu, M. Structures and Polymorphic Interactions of Two Heptad-Repeat Regions of the SARS Virus S2 Protein.
Structure 14, 889-899.
Bussell, R Jr, Ramlall, T F, Eliezer, D. Helix Periodicity, Topology and Dynamics of Membrane-Associated alpha-Synuclein. Prot Sci (2005) 14, 862872.
Eliezer, D, Barré, P, Kobaslija, M, Chan, D, Li, X, Heend, L. Residual structure in the repeat domain of tau: Echoes of microtubule binding and paired
helical filament formation. Biochemistry (2005) 44, 1026-1036.
Naik, M.T., Lee, H., Bracken, C. and Breslow, E., (2005) NMR Investigation of Main-Chain Dynamics of the H80E Mutant of Bovine Neurophysin-I:
Demonstration of Dimerization-Induced Changes at the Hormone-Binding Site, Biochemistry 44, 11766-11776
Mukherjee, M., Dutta, K., White, M.A., Cowburn, D., Fox, R.O. (2006) "NMR solution structure and backbone dynamics of domain III of the E protein of
tick-borne Langat flavivirus, suggests a potential site for molecular recognition" Protein Science 15 1342-1355.
Ferrage, F., Pelepussey P., Cowburn D., Bodenhausen G. "Intra-residue dipolar cross-relaxation rates between 13Ca and 13C' as a measure of
internal dynamics in proteins by NMR Spectroscopy" J. Am. Chem. Soc. 128, 11072-11078.
Salmon, J.B., Dubrocq, C., Tabeling, P., Charier, S., Alcor, D., Jullien, L., Ferrage, F. (2005) An Approach To Extract Rate Constants from ReactionDiffusion Dynamics in a Microchannel Anal. Chem. 77, 3417-3424
© NYSBC 7 Sep 2006
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Scientific Focus Areas,
NYSBC NMR affiliates and staff
Chemical
Physics
Methodology
Isotope
Labeling
Structures
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Dynamics
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Membrane
Proteins
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Complexes
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© NYSBC 7 Sep 2006
In cell
Protein
Expression
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Structures
free
Langat d3
CrkII-cSH3
N-terminal
Rhomboid
Protein Science, 15, 1342, 2006
Biochemistry, 45, 8874, 2006
J. Mol. Bio., 2006
bound
BPTF PHD
3+1 Human telomeric repeat
Nature, 442, 91, 2006
J. Am. Chem. Soc., 127, 17277, 2005
FADD
SWIRM
Vts1 SAM
DNA quadruplex
Mol. Cell 22, 599, 2006
Nat. Str. Mol. Bio., 12, 1078, 2005
J. Mol. Bio., 356, 1065, 2006
PNAS, 102, 634, 2005
© NYSBC 7 Sep 2006
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Dipolar Cross-relaxation rates between Cα and C’
Figure 5 Three typical buildup curves of the symmetrical
reconversion ratio of eq 1, with their best-fit curves and the
structure of ubiquitin. Filled red circles and the solid red line
correspond to the C'C pair of glutamine Q31 observed
through the NHN pair of aspartate D32 ( CR = (10.9 ± 0.12) ×
10-2 s-1). Blue crosses and the dashed blue line correspond to
the C'C pair of leucine L8 observed through the signal of
threonine T9 ( CR = (9.07 ± 0.09) × 10-2 s-1). Filled green
squares and the dotted green line correspond to the C C'
pair of arginine R74 observed through the signal of glycine
G75 ( CR = (2.33 ± 0.02) × 10-2 s-1). The structure of
ubiquitin39 was generated with MOLMOL.
Figure 7 Orange dots: correlation of experimental order
parameters S2(C'Cα) = (C'Cα)exp/g(C'Cα)rigid with
experimental order parameters S2(NHN) derived from 15N
relaxation rates. The four points circled in red represent
residues for which the extended model-free approach41 was
used to fit 15N relaxation data. The curves show the
theoretical correlation for the three-dimensional Gaussian
amplitude fluctuation (3D GAF) model with amplitudes σα
=σβ=κσγ (where σγ corresponds to fluctuations about the
Cαi-1Cαi vectors) for = 0, 0.5, 1, and 2 (red, light blue, dark
blue, and purple curves). The green diamonds (which are
almost exactly on the red curve) represents S2{ CR} = aS2{
(C'Cα)} + bS2{ (NC'NCα)}/(a + b) for very anisotropic local
motions (κ= 0, i.e., σα = σβ = 0) for weights a = 1 and b = 0.12.
JACS, 128, 11072, 2006
Dynamics Studies of FlaviVirus LANGAT domain III
Figure 5. Plot showing slow motion. (A) Residues showing Rex value (> 1.5 s-1) obtained from and R’ex analysis are painted (as green) on the
ribbon plot of the lowest-energy LGT-E-D3 NMR structure. Residues showing Rex value (B) which do not make any contact with E-D1 and
E-D2 in TBE are shown in green and those in contact with E-D1 (magenta) and E-D2 (blue) domains in TBE are shown in yellow on the
surface plot of the lowest-energy structure of LGT-E-D3 (SWISS-PROT). Contact regions were determined from the crystal structure of
TBE which bears 90% similarity to LGT, (C) which are in contact with two adjacent monomer unit (shown in magenta and blue) of LGTE-D3 pentamer crystal structure are shown in yellow and which do not show any contact are shown in green. (D) Surface plot of LGT-E-D3
showing residues that show Rex in LGT-E-D3 NMR analysis (green) and residues for which mutation studies have been done (red) in TBE,
LI, JE, WN and DEN-2 and residues for which mutational studies have been done and also happen to show slow dynamics (yellow).
© NYSBC 7 Sep 2006
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Dynamics Studies of Glutamate receptor Bound to Glutamate and
AMPA
Figure 5 Chemical shift perturbations. (a) A 1H 15N TROSY correlation
spectrum is shown for glutamate-bound GluR2 S1S2. (b) Chemical shift
perturbations, , are shown for AMPA- (red) and quisqualate-bound
(black) GluR2 S1S2, compared to the glutamate-bound complex, as a
function of the linear sequence of GluR2 S1S2. GT indicates the linker
region between S1 and S2. (c) Chemical shift perturbations of AMPAbound, compared to glutamate-bound, GluR2 S1S2 mapped onto a
ribbon diagram of glutamate-bound GluR2 S1S2 (pdb 1ftj, protomer a).
The peptide flip of GluR2 S1S2 is shown in the unflipped conformation.
Figure 9 Difference in Rex between glutamate- and AMPA-bound
GluR2 S1S2 mapped onto the glutamate-bound GluR2 S1S2 structure
(pdb 1ftj). Gray indicates amino acids that were not analyzed.
Differences are color-coded from white, 0 s-1, to red, ~10 s-1. The
potential subdomain between the loop containing Val683 and the
peptide flip is circled.
Biochemistry, 44, 3410, 2005
Structure and Dynamics of N-terminal domain of Rhomboid
Figure 8. NRho shows extensive
conformational flexibility for both the
backbone and the sidechains on the
μs-ms timescale. (a) Rex values at 600
MHz for NRho obtained from an
analysis of backbone R1, R2 and 1HN{15N} NOE data at 600 and 800 MHz
using the Lipari-Szabo model-free
formalism. Errors in the Rex values
are indicated by red risers. Rex values
scale as the square of the static
magnetic field. (b) Fits of the Ala3
(black) and Leu7 (red) Cmethyl-Cnext
zero-quantum coherences (ZQ,
experimental points: open circles;
theoretical curve: dotted line) and
double-quantum coherences (DQ,
experimental points: filled circles;
theoretical curve: solid line) to
Equation 1. Errors in the
experimental data points (Ala3: black,
Leu7: red) are indicated by the error
bars at the top right hand corner of
the figure.
Ghose et al, in press
Figure 9. Residues that display dynamics on the μs-ms timescale map on to a continuous surface. (a) Residues that display
large Rex values (> 5 s-1), as obtained from an analysis of backbone R1, R2 and 1HN−{15N} NOE data at 600 and 800 MHz
using the Lipari-Szabo model-free formalism, are shaded red. Backbone relaxation data corresponding to Ser35 and Gly36
could not be analyzed accurately due to large R2 values, these residues are shaded blue. Ala3 and Leu7 sidechains that
were shown to display slow dynamics as determined from multiple-quantum experiments involving methyl groups, are
displayed and shaded green. (b) The residues that display slow dynamics map onto a continuous surface contiguous to that
implicated in membrane-interaction. The shading scheme is the same as in (a) except in the case Leu7 that shows slow
dynamics both in the backbone and sidechain regions and is shaded yellow. (c) Residues that interact with C16PN
liposomes and display slow μs-ms timescale motion are shown in red. Residues that interact with C16PN liposomes but are
not significantly dynamic on the slow timescale are shown in yellow. Additional residues that are dynamic on the slow
timescale are shown in blue.
© NYSBC 7 Sep 2006
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