Improved transfer of two-dimensional crystals from the air/water interface to specimen support grids for high-resolution analysis by electron microscopy.
Electron crystallographic analysis of two-dimensional crystals grown on lipid layers at the air/water interface has been limited by loss or damage during transfer of the crystals to an electron microscope support grid. Two methods of transfer are described which are applicable on a small scale (10 microliters of protein solution) and which give greatly improved results for streptavidin crystals on biotinylated lipid layers. In the first method, a hydrophobic grid surface was produced by coating a carbon support film with a thin layer of SiO2, followed by alkylation with dimethyloctadecylchlorosilane. The transfer efficiency of protein crystals approached 50% coverage of the alkylated grid surface. The degree of order of crystals transferred to the alkylated grid surface and preserved in negative stain was significantly improved over that of crystals transferred directly to a carbon support film. In the second method, crystals at the air/water interface were transferred to a holey carbon support film. The efficiency of transfer across the holes was virtually 100% as nearly every hole was completely covered with crystals. After preservation of the crystals in 1% glucose and cooling to liquid nitrogen temperature, electron diffraction was obtained that extended to 1/2.8 A-1 resolution. This demonstrates that two-dimensional crystals grown on lipid layers at the air/water interface can be sufficiently well-ordered, even after transfer to a support grid, to yield high-resolution structural information.
Beckman Laboratories, Fairchild Center, Department of Cell Biology, Stanford University, School of Medicine, CA 94305.
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Kubalek
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Kornberg
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Darst
D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure*; D003460:Crystallization; D003461:Crystallography / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D019809:Streptavidin
D001426:Bacterial Proteins; D019809:Streptavidin