Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures
| Autor: | Jan Dohnálek, Vitaly Polovinkin, Krishna Khakurel, Janos Hajdu, Borislav Angelov, Bohdan Schneider, Jakob Andreasson, Michal Babiak |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Materials science
focused ion beam milling Ion beam Analytical chemistry Physics::Optics Materialkemi 010402 general chemistry complex mixtures 01 natural sciences Focused ion beam General Biochemistry Genetics and Molecular Biology Quantitative Biology::Subcellular Processes Crystal 03 medical and health sciences Structural Biology Materials Chemistry 030304 developmental biology Strukturbiologi Quantitative Biology::Biomolecules 0303 health sciences Electron crystallography lipidic cubic phases digestive oral and skin physiology technology industry and agriculture food and beverages Mesophase Research Papers 0104 chemical sciences Condensed Matter::Soft Condensed Matter membrane protein crystals grown in meso Lamella (surface anatomy) Electron diffraction Chemical engineering biological sciences electron diffraction Protein crystallization lamella preparation |
| Zdroj: | Journal of Applied Crystallography |
| Popis: | Electron diffraction experiments on crystals of membrane proteins grown in lipidic mesophases have not been possible owing to a thick layer of viscous crystallization medium around the crystals. Here it is shown that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can remove the viscous layer, and high-quality electron diffraction on a FIB-milled lamella of a bacteriorhodopsin 3D crystal is demonstrated. Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å−2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media. |
| Databáze: | OpenAIRE |
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