Journal Article

PUBDB-2024-01055

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins

Niebling, S.CFEL*Extern*EMBL* ; Veith, K.Extern* ; Vollmer, B.CSSB* ; Lizarrondo, J.CSSB*EMBL* ; Burastero, O.Extern*CSSB* ; Schiller, J.CSSB*EMBL* ; Struve Garcia, A.CSSB* ; Lewe, P.CSSB* ; Seuring, C.CFEL*CSSB*XFEL.EU* ; Witt, S.CSSB* ; García-Alai, M. (Corresponding author)

2022
Frontiers Lausanne

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Please use a persistent id in citations: doi:10.3389/fmolb.2022.882288  doi:10.3204/PUBDB-2024-01055

Abstract: Successful sample preparation is the foundation to any structural biology technique. Membrane proteins are of particular interest as these are important targets for drug design, but also notoriously difficult to work with. For electron cryo-microscopy (cryo-EM), the biophysical characterization of sample purity, homogeneity, and integrity as well as biochemical activity is the prerequisite for the preparation of good quality cryo-EM grids as these factors impact the result of the computational reconstruction. Here, we present a quality control pipeline prior to single particle cryo-EM grid preparation using a combination of biophysical techniques to address the integrity, purity, and oligomeric states of membrane proteins and its complexes to enable reproducible conditions for sample vitrification. Differential scanning fluorimetry following the intrinsic protein fluorescence (nDSF) is used for optimizing buffer and detergent conditions, whereas mass photometry and dynamic light scattering are used to assess aggregation behavior, reconstitution efficiency, and oligomerization. The data collected on nDSF and mass photometry instruments can be analyzed with web servers publicly available at spc.embl-hamburg.de. Case studies to optimize conditions prior to cryo-EM sample preparation of membrane proteins present an example quality assessment to corroborate the usefulness of our pipeline.

Note: Part of this work was performed at the Cryo-EMFacility at CSSB, supported by the UHH and DFG grantnumbers (INST 152/772-1|152/774-1|152/775-1|152/776-1|152/777-1 FUGG)

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Contributing Institute(s):

1. CSSB - Core Facility - Protein Production (CSSB-CF-PP)
2. CSSB - Leibniz-Institut für Experimentelle Virologie (LIV) - Kay Grünewald (CSSB-LIV-KG)
3. CSSB-CF-SPC (CSSB-CF-SPC)
4. CSSB-CF-CRYO (CSSB-CF-CRYO)

Research Program(s):

1. 899 - ohne Topic (POF4-899) (POF4-899)

Experiment(s):

1. No specific instrument

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