The cryogenic temperatures at which dynamic nuclear polarization (DNP) solid-state NMR experiments need to be carried out cause line-broadening, an effect that is especially detrimental for crowded protein spectra. By increasing the magnetic field strength from 600 to 800 MHz, the resolution of DNP spectra of type III secretion needles (T3SS) could be improved by 22 %, indicating that inhomogeneous broadening is not the dominant effect that limits the resolution of T3SS needles under DNP conditions. The outstanding spectral resolution of this system under DNP conditions can be attributed to its low overall flexibility

Baldus, Marc

Becker, Stefan

Chevelkov, Veniamin

Fricke, Pascal

Giller, Karin

Lange, Adam

Mance, Deni

Utrecht University Repository

High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles

The cryogenic temperatures at which dynamic nuclear polarization (DNP) solid-state NMR experiments need to be carried out cause line-broadening, an effect that is especially detrimental for crowded protein spectra. By increasing the magnetic field strength from 600 to 800 MHz, the resolution of DNP spectra of type III secretion needles (T3SS) could be improved by 22 %, indicating that inhomogeneous broadening is not the dominant effect that limits the resolution of T3SS needles under DNP conditions. The outstanding spectral resolution of this system under DNP conditions can be attributed to its low overall flexibility

Fricke, P.

Mance, D.

Chevelkov, V.

Giller, K.

Becker, S.

Baldus, M.

Lange, A.

MPG.PuRe

1 Supplementary Material High Resolution Observed in 800 MHz DNP Spectra of Extremely Rigid Type III Secretion Needles Pascal Fricke1, Deni Mance2, Veniamin Chevelkov1, Karin Giller3, Stefan Becker3, Marc Baldus2, Adam Lange1,4* 1 Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany 2 NMR research group, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands 3 Department of NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany 4 Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany * alange@fmp-berlin.de     Fig. S1 13C-13C SPC-5 2D NMR spectrum of MxiH needles at 800 MHz, 95 K and 8 kHz MAS under MW irradiation. Some of the peaks are assigned. Note that the indirect dimension is a double quantum dimension.     2 Preparation of the MxiH DNP Sample  The DNP sample was produced by preparing a 14 mM AMUPol solution in a 70/30 (v/v) glycerol-d8/D2O mixture. To a protein pellet consisting of uniformly 13C- and 15N-labeled MxiH needles roughly the same volume of this matrix was added. Subsequently, the substances were thoroughly mixed using a vortexer. The mixture was then filled into a 3.2 mm sapphire rotor which was closed with a zirconia cap (Fig. S2). Protonated water was not added to the matrix as the pellet still contained residual water buffer from the protein expression and purification.   Fig. S2 Illustration of the production process of the MxiH DNP sample.    Fig. S3 Comparison of two 1H-13C 1D CP NMR spectra of MxiH needles at 800 MHz, 95 K and 8 kHz MAS. Under otherwise identical conditions, the blue spectrum was recorded with the microwaves (MW) turned off and the red spectrum was recorded with the microwaves turned on. The signals are about 21 times stronger in the spectrum with the microwaves turned on. 3     Preparation of the PrgI T3SS Needle Sample used for T1 Measurements Expression, purification and polymerization of wild-type PrgI needles were performed as described before (Loquet et al. 2011; Loquet et al. 2012). Isotope labeling was achieved using 15NH4Cl as nitrogen source and D-[2-13C]-glucose as carbon source during expression. Approximately 15 mg of [2-13C]-glucose-, uniformly-15N-labeled protein was packed into a 3.2 mm rotor.  Solid-State NMR Experiments for Determination of T1 times of PrgI T3SS Needles Solid-state NMR experiments were conducted on an 800 MHz (1H Larmor frequency) NMR spectrometer (Bruker Biospin, Germany) equipped with a (1H, 13C, 15N) triple-resonance 3.2 mm probe. The sample was spun at 20 kHz. The effective sample temperature was 3±3 °C as measured by the temperature-dependent water proton resonance relative to an internal DSS reference (Boeckmann et al. 2009). Chemical shift referencing was achieved using the internal DSS reference. The longitudinal relaxation time was measured according to the pulse sequence introduced by Emsley and coworkers (Giraud et al. 2004) using a 15N-13CA 2D correlation spectrum to obtain residue specific information. Twelve spectra were recorded with 15N relaxation delays of 0, 5, 9, 17.5, 0, 33, 0, 60, 0, 110, 110, 0 seconds. All spectra were recorded with 64 scans, except the spectra with the relaxation delay of 110 seconds, which were recorded with 16 scans each.  During the direct (13C) and indirect (15N) isotropic chemical shift evolution periods, SPINAL-64 (Fung et al. 2000) heteronuclear decoupling was applied on the 1H channel with an RF field strength of 78 kHz. WALTZ-64 (Shaka et al. 1983) decoupling with an RF field strength of 2.5 kHz was applied on the 15N channel during the 13C detection period to remove heteronuclear scalar couplings. A hard 180° pulse was applied on the 13C channel to remove scalar couplings during the 15N indirect evolution period. The 13C and 15N acquisition times were 22 ms and 14 ms, respectively.   References Boeckmann A, Gardiennet C, Verel R, Hunkeler A, Loquet A, Pintacuda G, Emsley L, Meier BH, Lesage A (2009) Characterization of different water pools in solid-state NMR protein samples. J Biomol NMR 45:319-327 doi:10.1007/s10858-009-9374-3 Fung BM, Khitrin AK, Ermolaev K (2000) An improved broadband decoupling sequence for liquid crystals and solids. J Magn Reson 142:97-101 doi:10.1006/jmre.1999.1896 Giraud N, Bockmann A, Lesage A, Penin F, Blackledge M, Emsley L (2004) Site-specific backbone dynamics from a crystalline protein by solid-state NMR spectroscopy. J Am Chem Soc 126:11422-11423 Loquet A, Lv G, Giller K, Becker S, Lange A (2011) 13C spin dilution for simplified and complete solid-state NMR resonance assignment of insoluble biological assemblies. J Am Chem Soc 133:4722-4725 doi:10.1021/ja200066s Loquet A, Sgourakis NG, Gupta R, Giller K, Riedel D, Goosmann C, Griesinger C, Kolbe M, Baker D, Becker S, Lange A (2012) Atomic model of the type III secretion system needle. Nature 486:276-279 doi:10.1038/nature11079 Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broad-band decoupling - WALTZ-16. J Magn Reson 52:335-338 doi:10.1016/0022-2364(83)90207-x   

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High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles.

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High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles

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