Project description:Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air-water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.5 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including a smaller amount of protein required and avoiding protein denaturation at the air-water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the nonheme diiron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.
Project description:The fast development of single-particle cryogenic electron microscopy (cryo-EM) has made it more feasible to obtain the 3D structure of well-behaved macromolecules with a molecular weight higher than 300 kDa at ~3 Å resolution. However, it remains a challenge to obtain the high-resolution structures of molecules smaller than 200 kDa using single-particle cryo-EM. In this work, we apply the Cs-corrector-VPP-coupled cryo-EM to study the 52 kDa streptavidin (SA) protein supported on a thin layer of graphene and embedded in vitreous ice. We are able to solve both the apo-SA and biotin-bound SA structures at near-atomic resolution using single-particle cryo-EM. We demonstrate that the method has the potential to determine the structures of molecules as small as 39 kDa.
Project description:For cryo-EM structural studies, we seek to image membrane proteins as single particles embedded in proteoliposomes. One technical difficulty has been the low density of liposomes that can be trapped in the approximately 100nm ice layer that spans holes in the perforated carbon support film of EM grids. Inspired by the use of two-dimensional (2D) streptavidin crystals as an affinity surface for biotinylated DNA (Crucifix et al., 2004), we propose to use the crystals to tether liposomes doped with biotinylated lipids. The 2D crystal image also serves as a calibration of the image formation process, providing an absolute conversion from electrostatic potentials in the specimen to the EM image intensity, and serving as a quality control of acquired cryo-EM images. We were able to grow streptavidin crystals covering more than 90% of the holes in an EM grid, and which remained stable even under negative stain. The liposome density in the resulting cryo-EM sample was uniform and high due to the high-affinity binding of biotin to streptavidin. Using computational methods, the 2D crystal background can be removed from images without noticeable effect on image properties.
Project description:Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5Å or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5Å, while the resolution of the map reached 4.0Å for the same number of particles, when the estimated resolution of streptavidin crystal was 4Å or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6Å, a marked improvement over the value of 3.9Å obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.
Project description:Setd2 methylate the nucleosome to form H3K36me3. Here we utilized the Cryo-EM to elucidate the structure of SETD2/Set2 bound with nucleosomes. Through this structure analysis, we found that histone H1 may interfere the enzymatic activity of SETD2/Set2 by inhibiting their binding affinity.
Project description:High-quality specimen preparation plays a crucial role in cryo-electron microscopy (cryo-EM) structural analysis. In this study, we have developed a reliable and convenient technique called the graphene sandwich method for preparing cryo-EM specimens. This method involves using two layers of graphene films that enclose macromolecules on both sides, allowing for an appropriate ice thickness for cryo-EM analysis. The graphene sandwich helps to mitigate beam-induced charging effect and reduce particle motion compared to specimens prepared using the traditional method with graphene support on only one side, therefore improving the cryo-EM data quality. These advancements may open new opportunities to expand the use of graphene in the field of biological electron microscopy.
Project description:The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for ?-galactosidase bound to the inhibitor phenylethyl ?-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ? 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.
Project description:Parkinson's disease is a progressive neuropathological disorder that belongs to the class of synucleinopathies, in which the protein alpha-synuclein is found at abnormally high concentrations in affected neurons. Its hallmark are intracellular inclusions called Lewy bodies and Lewy neurites. We here report the structure of cytotoxic alpha-synuclein fibrils (residues 1-121), determined by cryo-electron microscopy at a resolution of 3.4 Å. Two protofilaments form a polar fibril composed of staggered ?-strands. The backbone of residues 38 to 95, including the fibril core and the non-amyloid component region, are well resolved in the EM map. Residues 50-57, containing three of the mutation sites associated with familial synucleinopathies, form the interface between the two protofilaments and contribute to fibril stability. A hydrophobic cleft at one end of the fibril may have implications for fibril elongation, and invites for the design of molecules for diagnosis and treatment of synucleinopathies.
Project description:Chaperonins play an important role in folding newly synthesized or translocated proteins in all organisms. The bacterial chaperonin GroEL has served as a model system for the understanding of these proteins. In comparison, its human homolog, known as mitochondrial heat shock protein family member D1 (HSPD1) is poorly understood. Here, we present the structure of HSPD1 in the apo state determined by cryo-electron microscopy (cryo-EM). Unlike GroEL, HSPD1 forms mostly single ring assemblies in the absence of co-chaperonin (HSPE1). Comparison with GroEL shows a rotation and increased flexibility of the apical domain. Together with published structures of the HSPD1/HSPE1 co-chaperonin complex, this work gives insight into the structural changes that occur during the catalytic cycle. This new understanding of HSPD1 structure and its rearrangements upon complex formation may provide new insights for the development of HSPD1-targeting treatments against a diverse range of diseases including glioblastoma.