Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography
| Autor: | David I. Stuart, Gwyndaf Evans, Adam D. Crawshaw, Anna J. Warren, Emma V Beale, James Beilsten-Edmands, Geoff Sutton, José Trincão |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Diffraction
Materials science Scanning electron microscope Analytical chemistry microfocus X-ray diffraction engineering.material Biochemistry 03 medical and health sciences 0302 clinical medicine macromolecular crystallography Microscopy structural biology General Materials Science 030304 developmental biology 0303 health sciences Crystallography visualization tools Resolution (electron density) Diamond General Chemistry Condensed Matter Physics Research Papers VMXm beamline cryoEM Nanocrystal Beamline QD901-999 radiation damage engineering Protein crystallization 030217 neurology & neurosurgery scanning electron microscopy |
| Zdroj: | IUCrJ IUCrJ, Vol 7, Iss 3, Pp 500-508 (2020) |
| ISSN: | 2052-2525 |
| Popis: | Protein crystals exposed to a scanning electron microscope beam were analysed using X-ray diffraction to assess the impact of electrons on crystal quality. These experiments support the use of a scanning electron microscope as an imaging tool for locating and centring microcrystals with a view to implement this technology on the VMXm beamline at Diamond Light Source. 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|