NMR Facility Operations at NYSBC

Albert Einstein College of Medicine of Yeshiva University City University of New York Columbia University Memorial Sloan Kettering Cancer Center Mount Sinai School of Medicine New York University Rockefeller University State University of New York Wadsworth Center, NYS Dept. Health/HRI Weill Cornell Medical College of Cornell University

© NYSBC 7 Sep 2006 2 NYSBC operates cryo-electron microscopy resources, two beam lines at NSLS/BNL, and a large NMR facility. There are investigator- headed 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

0.1% 2mM Sucrose Spectrometer Probe Ethylbenzene Anomeric 1H S/N S/N 900 #1 cp TCI 8707:1 1006:1 900 US2 #2 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

90% B1 LowerTemp. Probes Tuning Fields, kHz. S/N(1,2) Homogeneity Range, oC(3) 750 MHz Range, MHz 1H/13C/15N Volume 10kHz/15kHz

HXY X: 150-200 13C 160:1 120/50/50 35-40 μl -40/-30 wide bore Y:70-130 15N: 30:1

HFX 13C 240:1 X:35-225 120/50/50 35-40 μl -40/-30 wide bore 15N: 30:1

HCN 13C 180:1 standard narrowband 120/50/50 35-40 μl -15/-5 15N: 30:1 bore HRMAS 90o pulse: narrowband 1H : 250:1 35-40 μl n.a. HCND 6μs/8μs/15μs

(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.

Heteronuclear spin-echo difference test is run on a sample The lineshape stability test is run on a non-spinning containing 500 mM Sucrose in 100% D2O. In this test the sample of 0.3% CHCl3 in Acetone-D6. The lineshape is 12C center-band of the sucrose anomeric protons is described by the hump numbers (linewidth at 0.55%/0.11% suppressed in two scans, below the intensity of natural of the carbon satellite peaks and half height of the main abundance 13C side-bands in a series of 20 consecutive chloroform peak). In this test the hump numbers should experiments. 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

Red surface are at the 5 G contour

© NYSBC 7 Sep 2006 6 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).

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(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. 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Chemical Isotope In cell Protein Physics Labeling Expression Methodology

Structures * * *

Dynamics * *

Membrane Proteins * * * *

Complexes * * * *

free

bound N-terminal Langat d3 CrkII-cSH3 Rhomboid BPTF PHD 3+1 Human telomeric repeat Protein Science, 15, 1342, 2006 Biochemistry, 45, 8874, 2006 J. Mol. Bio., 2006 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

Figure 7 Orange dots: correlation of experimental order 2 parameters S (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 Figure 5 Three typical buildup curves of the symmetrical used to fit 15N relaxation data. The curves show the reconversion ratio of eq 1, with their best-fit curves and the theoretical correlation for the three-dimensional Gaussian structure of ubiquitin. Filled red circles and the solid red line amplitude fluctuation (3D GAF) model with amplitudes σα correspond to the C'C pair of glutamine Q31 observed =σβ=κσγ (where σγ corresponds to fluctuations about the N α α through the NH pair of aspartate D32 ( CR = (10.9 ± 0.12) × C i-1C i vectors) for = 0, 0.5, 1, and 2 (red, light blue, dark 10-2 s-1). Blue crosses and the dashed blue line correspond to blue, and purple curves). The green diamonds (which are 2 2 the C'C pair of leucine L8 observed through the signal of almost exactly on the red curve) represents S { CR} = aS { -2 -1 2 threonine T9 ( CR = (9.07 ± 0.09) × 10 s ). Filled green (C'Cα)} + bS { (NC'NCα)}/(a + b) for very anisotropic local squares and the dotted green line correspond to the C C' motions (κ= 0, i.e., σα = σβ = 0) for weights a = 1 and b = - pair of arginine R74 observed through the signal of glycine 0.12. -2 -1 G75 ( CR = (2.33 ± 0.02) × 10 s ). The structure of ubiquitin39 was generated with MOLMOL. JACS, 128, 11072, 2006

Dynamics Studies of FlaviVirus LANGAT domain III

-1 ’ Figure 5. Plot showing slow motion. (A) Residues showing Rex value (> 1.5 s ) 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 LGT- E-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 12 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 Figure 9 Difference in Rex between glutamate- and AMPA-bound (black) GluR2 S1S2, compared to the glutamate-bound complex, as a GluR2 S1S2 mapped onto the glutamate-bound GluR2 S1S2 structure function of the linear sequence of GluR2 S1S2. GT indicates the linker (pdb 1ftj). Gray indicates amino acids that were not analyzed. -1 -1 region between S1 and S2. (c) Chemical shift perturbations of AMPA- Differences are color-coded from white, 0 s , to red, ~10 s . The bound, compared to glutamate-bound, GluR2 S1S2 mapped onto a potential subdomain between the loop containing Val683 and the ribbon diagram of glutamate-bound GluR2 S1S2 (pdb 1ftj, protomer a). peptide flip is circled. The peptide flip of GluR2 S1S2 is shown in the unflipped conformation. 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 1 N analysis of backbone R1, R2 and H - {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, Figure 9. Residues that display dynamics on the μs-ms timescale map on to a continuous surface. (a) Residues that display -1 1 N 15 experimental points: filled circles; large Rex values (> 5 s ), as obtained from an analysis of backbone R1, R2 and H −{ N} NOE data at 600 and 800 MHz theoretical curve: solid line) to 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 R values, these residues are shaded blue. Ala3 and Leu7 sidechains that Equation 1. Errors in the 2 were shown to display slow dynamics as determined from multiple-quantum experiments involving methyl groups, are experimental data points (Ala3: black, displayed and shaded green. (b) The residues that display slow dynamics map onto a continuous surface contiguous to that Leu7: red) are indicated by the error implicated in membrane-interaction. The shading scheme is the same as in (a) except in the case Leu7 that shows slow bars at the top right hand corner of dynamics both in the backbone and sidechain regions and is shaded yellow. (c) Residues that interact with C16PN the figure. 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 Ghose et al, in press 13