Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins
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 s...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2022.882288/full |
| _version_ | 1811335205863030784 |
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| author | Stephan Niebling Stephan Niebling Katharina Veith Benjamin Vollmer Javier Lizarrondo Osvaldo Burastero Osvaldo Burastero Janina Schiller Angelica Struve García Angelica Struve García Philipp Lewe Carolin Seuring Susanne Witt María García-Alai María García-Alai |
| author_facet | Stephan Niebling Stephan Niebling Katharina Veith Benjamin Vollmer Javier Lizarrondo Osvaldo Burastero Osvaldo Burastero Janina Schiller Angelica Struve García Angelica Struve García Philipp Lewe Carolin Seuring Susanne Witt María García-Alai María García-Alai |
| author_sort | Stephan Niebling |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T17:20:41Z |
| format | Article |
| id | doaj.art-b0f17dbc4aac4036962350cb153663d5 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-04-13T17:20:41Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-b0f17dbc4aac4036962350cb153663d52022-12-22T02:37:59ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2022-06-01910.3389/fmolb.2022.882288882288Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane ProteinsStephan Niebling0Stephan Niebling1Katharina Veith2Benjamin Vollmer3Javier Lizarrondo4Osvaldo Burastero5Osvaldo Burastero6Janina Schiller7Angelica Struve García8Angelica Struve García9Philipp Lewe10Carolin Seuring11Susanne Witt12María García-Alai13María García-Alai14European Molecular Biology Laboratory Hamburg, Hamburg, GermanyCentre for Structural Systems Biology (CSSB), Hamburg, GermanyEuropean Molecular Biology Laboratory Hamburg, Hamburg, GermanyCentre for Structural Systems Biology (CSSB), Leibniz Institute of Virology (LIV), Hamburg, GermanyEuropean Molecular Biology Laboratory Hamburg, Hamburg, GermanyEuropean Molecular Biology Laboratory Hamburg, Hamburg, GermanyCentre for Structural Systems Biology (CSSB), Hamburg, GermanyEuropean Molecular Biology Laboratory Hamburg, Hamburg, GermanyEuropean Molecular Biology Laboratory Hamburg, Hamburg, GermanyCentre for Structural Systems Biology (CSSB), Hamburg, GermanyCentre for Structural Systems Biology (CSSB), University Medical Center Hamburg-Eppendorf, Hamburg, GermanyCentre for Structural Systems Biology (CSSB), Department of Chemistry, University of Hamburg, Hamburg, GermanyCentre for Structural Systems Biology (CSSB), University Medical Center Hamburg-Eppendorf, Hamburg, GermanyEuropean Molecular Biology Laboratory Hamburg, Hamburg, GermanyCentre for Structural Systems Biology (CSSB), Hamburg, GermanySuccessful 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.https://www.frontiersin.org/articles/10.3389/fmolb.2022.882288/fullmass photometrydifferential scanning fluorimetry (DSF)membrane proteinsdynamic light scatteringsample preparationelectron cryo-microscopy (cryo-EM) |
| spellingShingle | Stephan Niebling Stephan Niebling Katharina Veith Benjamin Vollmer Javier Lizarrondo Osvaldo Burastero Osvaldo Burastero Janina Schiller Angelica Struve García Angelica Struve García Philipp Lewe Carolin Seuring Susanne Witt María García-Alai María García-Alai Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins Frontiers in Molecular Biosciences mass photometry differential scanning fluorimetry (DSF) membrane proteins dynamic light scattering sample preparation electron cryo-microscopy (cryo-EM) |
| title | Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins |
| title_full | Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins |
| title_fullStr | Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins |
| title_full_unstemmed | Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins |
| title_short | Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins |
| title_sort | biophysical screening pipeline for cryo em grid preparation of membrane proteins |
| topic | mass photometry differential scanning fluorimetry (DSF) membrane proteins dynamic light scattering sample preparation electron cryo-microscopy (cryo-EM) |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2022.882288/full |
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