Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography
Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large...
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| Format: | Article |
| Language: | English |
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International Union of Crystallography
2020-05-01
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| Series: | IUCrJ |
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| Online Access: | http://scripts.iucr.org/cgi-bin/paper?S2052252520003875 |
| _version_ | 1828130343844052992 |
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| author | Emma V. Beale Anna J. Warren José Trincão James Beilsten-Edmands Adam D. Crawshaw Geoff Sutton David Stuart Gwyndaf Evans |
| author_facet | Emma V. Beale Anna J. Warren José Trincão James Beilsten-Edmands Adam D. Crawshaw Geoff Sutton David Stuart Gwyndaf Evans |
| author_sort | Emma V. Beale |
| collection | DOAJ |
| description | Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline. |
| first_indexed | 2024-04-11T16:32:48Z |
| format | Article |
| id | doaj.art-97953cece9ad46f69d67226b4d3ea88b |
| institution | Directory Open Access Journal |
| issn | 2052-2525 |
| language | English |
| last_indexed | 2024-04-11T16:32:48Z |
| publishDate | 2020-05-01 |
| publisher | International Union of Crystallography |
| record_format | Article |
| series | IUCrJ |
| spelling | doaj.art-97953cece9ad46f69d67226b4d3ea88b2022-12-22T04:13:58ZengInternational Union of CrystallographyIUCrJ2052-25252020-05-017350050810.1107/S2052252520003875jt5045Scanning electron microscopy as a method for sample visualization in protein X-ray crystallographyEmma V. Beale0Anna J. Warren1José Trincão2James Beilsten-Edmands3Adam D. Crawshaw4Geoff Sutton5David Stuart6Gwyndaf Evans7Life Science, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UKLife Science, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UKLife Science, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UKLife Science, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UKLife Science, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UKDivision of Structural Biology, University of Oxford, Wellcome Centre for Human Genetics, Oxford, Oxfordshire OX3 7BN, UKLife Science, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UKLife Science, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UKDeveloping methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.http://scripts.iucr.org/cgi-bin/paper?S2052252520003875microfocus x-ray diffractionvmxm beamlinemacromolecular crystallographycryoemstructural biologyradiation damagescanning electron microscopyvisualization tools |
| spellingShingle | Emma V. Beale Anna J. Warren José Trincão James Beilsten-Edmands Adam D. Crawshaw Geoff Sutton David Stuart Gwyndaf Evans Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography IUCrJ microfocus x-ray diffraction vmxm beamline macromolecular crystallography cryoem structural biology radiation damage scanning electron microscopy visualization tools |
| title | Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography |
| title_full | Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography |
| title_fullStr | Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography |
| title_full_unstemmed | Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography |
| title_short | Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography |
| title_sort | scanning electron microscopy as a method for sample visualization in protein x ray crystallography |
| topic | microfocus x-ray diffraction vmxm beamline macromolecular crystallography cryoem structural biology radiation damage scanning electron microscopy visualization tools |
| url | http://scripts.iucr.org/cgi-bin/paper?S2052252520003875 |
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