Improved High-resolution cryo-EM Methodology

University Of Calif-Lawrenc Berkeley Lab, Berkeley CA

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Abstract

Core - Improved High-resolution cryo-EM Methodology Jamie Cate and Kenneth Downing PROJECT SUMMARY/ABSTRACT The capabilities of cryo-EM have begun to match or surpass those of X-ray crystallography in many areas of structural biology. However, there remain crucial bottlenecks in cryo-EM that hamper reconstructions of fragile complexes. The goal of this Core Project is to develop methods that will further enhance the capabilities of cryo-EM, in order to support the work of the Projects and Associated Projects of this Program. We address two key areas where single-particle cryo-EM requires further improvement. We will first address the cryo-preservation of delicate samples on cryo-EM grids. Many samples are subject to denaturation when exposed to the extensive air-water interface during blotting, or to a continuous carbon grid. To prepare specimens in a more structure-friendly way by avoiding specimen contact with the air-water interface, streptavidin monolayer crystals will be used as a specimen-support film for affinity-binding interactions. We will test a series of affinity adaptor and affinity tagging methods, to ensure uniform particle orientations on the streptavidin monolayer crystal lattice. These experiments will demonstrate the general utility of using streptavidin monolayer crystals as a support material. Second, we will develop means to correct beam-induced sample movement. When using direct electron detectors in ?movie? mode, the initial frames of exposures are presently discarded, although they potentially contain the highest resolution information. In subsequent frames, the high-resolution information is no longer available due to electron beam induced damage. We propose to use the image of the streptavidin support film as a fiducial, to better characterize the frame-to-frame motions that occur. We will use human 48S translation preinitiation complexes and Escherichia coli 70S ribosomes as test systems to validate the methods in these two aims. Once we have established these methods, they will be of wide use in the cryo-EM community, and should greatly expand the scope of cryo-EM into areas of structural biology that were previously inaccessible, or accessible only to x-ray crystallography.