Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cryo-EM with sub-1 A specimen movement.

ABSTRACT: Most information loss in cryogenic electron microscopy (cryo-EM) stems from particle movement during imaging, which remains poorly understood. We show that this movement is caused by buckling and subsequent deformation of the suspended ice, with a threshold that depends directly on the shape of the frozen water layer set by the support foil. We describe a specimen support design that eliminates buckling and reduces electron beam-induced particle movement to less than 1 angstrom. The design allows precise foil tracking during imaging with high-speed detectors, thereby lessening demands on cryostage precision and stability. It includes a maximal density of holes, which increases throughput in automated cryo-EM without degrading data quality. Movement-free imaging allows extrapolation to a three-dimensional map of the specimen at zero electron exposure, before the onset of radiation damage.

SUBMITTER: Naydenova K

PROVIDER: S-EPMC7116250 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Cryo-EM with sub-1 Å specimen movement.

Naydenova Katerina K Jia Peipei P Russo Christopher J CJ

Science (New York, N.Y.) 20201001 6513

Most information loss in cryogenic electron microscopy (cryo-EM) stems from particle movement during imaging, which remains poorly understood. We show that this movement is caused by buckling and subsequent deformation of the suspended ice, with a threshold that depends directly on the shape of the frozen water layer set by the support foil. We describe a specimen support design that eliminates buckling and reduces electron beam-induced particle movement to less than 1 angstrom. The design allow ...[more]

PMID: 33033219

Similar Datasets

Graphene sandwich-based biological specimen preparation for cryo-EM analysis.

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.

| S-EPMC10835136 | biostudies-literature

Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.

Project description:Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

| S-EPMC6889916 | biostudies-literature

Addressing preferred specimen orientation in single-particle cryo-EM through tilting.

Project description:We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes. We applied these methods to determine the structures at near-atomic resolution of the influenza hemagglutinin trimer, which adopts a highly preferred specimen orientation, and of ribosomal biogenesis intermediates, which adopt moderately preferred orientations.

| S-EPMC5533649 | biostudies-literature

Devitrification reduces beam-induced movement in cryo-EM.

Project description:As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent problems has been beam-induced movement, which occurs when the specimen is irradiated with high-energy electrons. Beam-induced movement results in image blurring and loss of high-resolution information. It is particularly severe for biological samples in unsupported thin films of vitreous water. By controlled devitrification of conventionally plunge-frozen samples, the suspended film of vitrified water was converted into cubic ice, a polycrystalline, mechanically stable solid. It is shown that compared with vitrified samples, devitrification reduces beam-induced movement in the first 5 e Å-2 of an exposure by a factor of ∼4, substantially enhancing the contribution of the initial, minimally damaged frames to a structure. A 3D apoferritin map reconstructed from the first frames of 20 000 particle images of devitrified samples resolved undamaged side chains. Devitrification of frozen-hydrated specimens helps to overcome beam-induced specimen motion in single-particle cryo-EM, as a further step towards realizing the full potential of cryo-EM for high-resolution structure determination.

| S-EPMC7924229 | biostudies-literature

Cryo-EM phase-plate images reveal unexpected levels of apparent specimen damage.

Project description:Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 Å resolution or higher. When we imaged apoF with a laser phase plate (LPP), however, we observed more severe particle-to-particle variation in the images than we had previously thought to exist. Similarly, we found that images of ribulose bisphosphate carboxylase/oxygenase (rubisco) also exhibited a much greater amount of heterogeneity than expected. By comparison to simulations of images, we verified that the heterogeneity is not explained by the known features of the LPP, shot noise, or differences in particle orientation. We also demonstrate that our specimens are comparable to those previously used in the literature, based on using the final-reconstruction resolution as the metric for evaluation. All of this leads us to the hypothesis that the heterogeneity is due to damage that has occurred either during purification of the specimen or during preparation of the grids. It is not, however, our goal to explain the causes of heterogeneity; rather, we report that using the LPP has made the apparent damage too obvious to be ignored. In hindsight, similar heterogeneity can be seen in images of apoF and the 20S proteasome which others had recorded with a Volta phase plate. We therefore conclude that the increased contrast of phase-plate images (at low spatial frequencies) should also make it possible to visualize, on a single-particle basis, various forms of biologically functional heterogeneity in structure that had previously gone unnoticed.

| S-EPMC11326166 | biostudies-literature

Towards sub-millisecond cryo-EM grid preparation.

Project description:Sample preparation is still a significant problem for many single particle cryo-EM workflows and our understanding and developments in the area lag behind that of image processing and microscope design. Over the last few years there has been growing evidence that many of the problems which occur during sample preparation are during the time the sample resides within the thin film created during the conventional blotting process. In parallel, faster grid preparation approaches have been developed for time-resolved cryo-EM experiments allowing for non-equilibrium intermediates to be captured on the ms timescale. Therefore, an important question is how fast can we prepare suitable grids for imaging by cryo-EM and how much does this mitigate the problems observed in sample preparation? Here we use a novel approach which has been developed for time-resolved studies to produce grids on an estimated sub-1 ms timescale. While the method comes with its own challenges, a 3.8 Å reconstruction of apoferritin prepared with the ultrafast method shows that good resolutions can be achieved. Although several orders of magnitude faster than conventional approaches we show using a ribosome sample, that interactions with the air-water interface cannot be avoided with preferred orientations still present. Therefore, the work shows that faster reactions can be captured but poses the question whether speed is the answer to problems with sample preparation.

| S-EPMC9642037 | biostudies-literature

Cryo-EM shows how dynactin recruits two dyneins for faster movement.

Project description:Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased towards recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two dyneins. We find that the shift towards a double dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a dynein tail-dynactin-BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein-dynactin transport machine.

| S-EPMC5988349 | biostudies-literature

Methods to account for movement and flexibility in cryo-EM data processing.

Project description:Recent advances in direct electron detectors and improved CMOS cameras have been accompanied by the development of a range of software to take advantage of the data they produce. In particular they allow for the correction of two types of motion in cryo electron microscopy samples: motion correction for movements of the sample particles in the ice, and differential masking to account for heterogeneity caused by flexibility within protein complexes. Here we provide several scripts that allow users to move between RELION and standalone motion correction and centring programs. We then compare the computational cost and improvements in data quality with each program. We also describe our masking procedures to account for conformational flexibility. For the different elements of this study we have used three samples; a high symmetry virus, flexible protein complex (∼1MDa) and a relatively small protein complex (∼550kDa), to benchmark four widely available motion correction packages. Using these as test cases we demonstrate how motion correction and differential masking, as well as an additional particle re-centring protocol can improve final reconstructions when used within the RELION image-processing package.

| S-EPMC4854228 | biostudies-literature

Cryo-EM structures of inactive and active GABAB receptor.

Project description:Metabotropic GABAB G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABAB receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of Gi1 protein at a resolution range of 2.8-3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of Gi1. We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABAB receptor.

| S-EPMC7343782 | biostudies-literature

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB2-AcpM2.

Project description:Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

| S-EPMC7305291 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data