ArticleFrontiers in molecular biosciences2022
Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins.
Article in Frontiers in molecular biosciences, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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13 citing papers in PubMed.
- Structural basis of phosphatidylserine enhancement of Gas6 ligand-induced Axl activation.bioRxiv : the preprint server for biology · 2026Article
- Clathrin adaptors drive phase separation in endocytosis and trafficking.Communications biology · 2026Article
- Kinase condensates enrich ATP and trigger autophosphorylation.Cell reports · 2026Article
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- Despite the odds: formation of the SARS-CoV-2 methylation complex.Nucleic acids research · 2024Article
- Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions.Nature communications · 2024Article
- Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1.Nature structural & molecular biology · 2024Article
- RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment.Journal of the American Chemical Society · 2023Article
- Diacylglycerol-dependent hexamers of the SNARE-assembling chaperone Munc13-1 cooperatively bind vesicles.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- BRCA2-HSF2BP oligomeric ring disassembly by BRME1 promotes homologous recombination.Science advances · 2023Article
- Raynals, an online tool for the analysis of dynamic light scattering.Acta crystallographica. Section D, Structural biology · 2023Article
- Mass spectrometry of intact membrane proteins: shifting towards a more native-like context.Essays in biochemistry · 2023Review
- Biophysical Characterization of Membrane Proteins.Methods in molecular biology (Clifton, N.J.) · 2023Article
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11 authors.
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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.
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