Ultrastable gold substrates for electron cryomicroscopy

Abstract

Despite recent advances, the structures of many proteins cannot be determined by electron cryomicroscopy because the individual proteins move during irradiation. This blurs the images so they cannot be aligned with each other to calculate a 3D density. Much of this movement stems from instabilities in the carbon substrates used to support frozen samples in the microscope. Here we demonstrate a new gold specimen support that nearly eliminates substrate motion during irradiation. This increases the sub-nanometer image contrast such that α-helices of individual proteins are resolved. With this improvement we determine the structure of apoferritin, a smooth, octahedral shell of α-helical subunits that is particularly difficult to solve by electron microscopy.This advance in substrate design will enable the solution of currently intractable protein structures.Recent developments in electron cryomicroscopy (cryo-EM) have allowed structure determination to near-atomic resolution for some macromolecular complexes (1-3). Still, many small and challenging structures cannot be determined by current cryo-EM methods to the resolutions required for accurate modeling of atomic structure. This is because electron micrograph quality still falls short of the physical limits imposed by radiation damage to the macromolecules (4-6). Reduced image quality likely has two primary origins: Inefficient detection of imaging electrons (6, 7), and motion and blurring of the particles during irradiation (6,(8)(9)(10)(11). Recent developments in electron detectors have addressed the first constraint (12,13), and have enabled the development of motion correction algorithms to ameliorate the effects of the second (14-16). Direct electron detectors can acquire images using fractions of the electron dose previously required, thus allowing the measurement of single molecule positions in time. This allows accurate tracking of large ensembles of particles, and offers a way to determine the physical origins of radiation-induced particle movement (14-17).Conventional amorphous carbon (am-C) substrates undergo bending and deformation during irradiation (9,11), which includes movement both parallel and perpendicular to the substrate plane. Incorporation of titanium-silicon, doped silicon carbide or graphene into substrate designs reduced radiation-induced specimen motion (17)(18)(19). Still these designs did not stop substrate movement and are challenging to manufacture and use. Here, we demonstrate a new cryo-EM support that nearly eliminates the radiation-induced deformation of thin, iceembedded specimens at cryogenic temperatures. This curtails the perpendicular and in-plane * To whom correspondence should be addressed; passmore@mrc-lmb.cam.ac.uk. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts components of motion during imaging, and thus improves image quality for all radiationsensitive cryogenic specimens.The support comprises a regular array of micron sized holes in a ≃500 Å thick foil ...

Opening of the human epithelial calcium channel TRPV6

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Structural Basis for Gating and Activation of RyR1

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