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ABSTRACT Solid-state NMR spectroscopy has been used successfully for characterizing the structure and dynamics of membrane proteins as well as their interactions with other proteins in lipid

bilayers. Such an environment is often necessary for achieving native-like structures. Sample preparation is the key to this success. Here we present a detailed description of a robust

protocol that results in high-quality membrane protein samples for both magic-angle spinning and oriented-sample solid-state NMR. The procedure is demonstrated using two proteins: CrgA (two

transmembrane helices) and Rv1861 (three transmembrane helices), both from _Mycobacterium tuberculosis_. The success of this procedure relies on two points. First, for samples for both types

of NMR experiment, the reconstitution of the protein from a detergent environment to an environment in which it is incorporated into liposomes results in 'complete' removal of

detergent. Second, for the oriented samples, proper dehydration followed by rehydration of the proteoliposomes is essential. By using this protocol, proteoliposome samples for magic-angle

spinning NMR and uniformly aligned samples (orientational mosaicity of <1°) for oriented-sample NMR can be obtained within 10 d. Access through your institution Buy or subscribe This is a

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RETRIEVE’ METHODOLOGY TO SIMULTANEOUSLY SOLVE CRYO-EM STRUCTURES OF MEMBRANE PROTEINS Article 06 January 2021 THE IMPORTANCE OF THE MEMBRANE FOR BIOPHYSICAL MEASUREMENTS Article 16 November

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spectroscopy sample preparation for helical integral membrane proteins. _J. Struct. Funct. Genomics_ 7, 51–64 (2006). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We

thank M.W. Davidson at NHMFL and C. Escobar at FSU, IMB, NHMFL for helping with photography. We also thank P.L. Gor'kov for his design of the OS sample holder and the sample transfer

base. This work was supported in part by the US National Institutes of Health (grants AI 074805, AI 073891 and AI 023007) and the US National Science Foundation (through Cooperative

Agreement 0654118 between the Division of Materials Research and the State of Florida). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Institute of Molecular Biophysics (IMB), Florida State

University (FSU), Tallahassee, Florida, USA Nabanita Das, Dylan T Murray & Timothy A Cross * National High Magnetic Field Laboratory (NMHFL), FSU, Tallahassee, Florida, USA Nabanita Das,

 Dylan T Murray & Timothy A Cross * Department of Chemistry and Biochemistry, FSU, Tallahassee, Florida, USA Timothy A Cross Authors * Nabanita Das View author publications You can also

search for this author inPubMed Google Scholar * Dylan T Murray View author publications You can also search for this author inPubMed Google Scholar * Timothy A Cross View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS N.D. and D.T.M. performed all the experiments such as membrane protein expression, purification,

solid-state NMR sample preparation and new method development. N.D. prepared all the figures. N.D. and T.A.C. wrote the manuscript, D.T.M. provided essential comments. All three authors

coordinated to complete this manuscript. CORRESPONDING AUTHOR Correspondence to Timothy A Cross. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests.

INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 12% (WT/VOL) SDS-PAGE OF CRGA AND RV1861 MEMBRANE PROTEIN EXPRESSION AND PURIFICATION STEPS. M: Molecular weight marker, L: Whole

cell lysate containing inclusion body and membrane fractions, FT: Flow through from nickel column, Washes: two to three consecutive washing steps, Elutions: Protein elution from nickel

column. Molecular weights of the proteins are shown by red color arrows. SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 12% (wt/vol) SDS-PAGE of CrgA and Rv1861 membrane protein expression

and purification steps. (PDF 3068 kb) SUPPLEMENTARY METHODS CrgA and Rv1861 membrane protein expression and purification; Reconstitution and OS sample preparation of 15N uniform labeled

gramicidin A protein in DMPC lipid bilayers. (PDF 208 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Das, N., Murray, D. & Cross, T. Lipid

bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples. _Nat Protoc_ 8, 2256–2270 (2013). https://doi.org/10.1038/nprot.2013.129 Download

citation * Published: 24 October 2013 * Issue Date: November 2013 * DOI: https://doi.org/10.1038/nprot.2013.129 SHARE THIS ARTICLE Anyone you share the following link with will be able to

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