Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multifunctional graphene supports for electron cryomicroscopy.

ABSTRACT: With recent technological advances, the atomic resolution structure of any purified biomolecular complex can, in principle, be determined by single-particle electron cryomicroscopy (cryoEM). In practice, the primary barrier to structure determination is the preparation of a frozen specimen suitable for high-resolution imaging. To address this, we present a multifunctional specimen support for cryoEM, comprising large-crystal monolayer graphene suspended across the surface of an ultrastable gold specimen support. Using a low-energy plasma surface modification system, we tune the surface of this support to the specimen by patterning a range of covalent functionalizations across the graphene layer on a single grid. This support design reduces specimen movement during imaging, improves image quality, and allows high-resolution structure determination with a minimum of material and data.

SUBMITTER: Naydenova K

PROVIDER: S-EPMC6575631 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Movies of horse spleen apoferritin on multifunctional ultrastable graphene supports for electron cryomicroscopy

Project description: Not available

| EMPIAR-10272 | biostudies-other

Integrated wafer-scale manufacturing of electron cryomicroscopy specimen supports.

Project description:We present a process for the manufacture of electron cryomicroscopy (cryoEM) specimen supports with an integrated foil-grid structure, using cryogenic vacuum evaporation (cryoEvap) and patterned electroplating on a silicon wafer substrate. The process is designed to produce a pattern of nanometre scale holes in a thin metal foil, which is attached to a pattern of micrometre scale grid bars that support it and allow handling of the millimetre scale device. All steps are carried out on a single 4 inch (100 mm) silicon wafer, without any need to handle individual grids during processing, and yield about 600 supports per wafer. The approach is generally applicable to the problem of creating a thin foil with nanometre scale features and a micrometre scale support structure; here it is used to make an all gold, HexAuFoil type design. It also allows for the addition of custom fiducial markers and patterns which aid in locating and identifying particular regions of a grid at several length scales: by eye, in an optical microscope, and in the electron microscope. Implemented at scale, this manufacturing process can supply ample grids to support the continued growth of cryoEM for determining the structure of biological molecules.

| S-EPMC8689146 | biostudies-literature

Charge accumulation in electron cryomicroscopy.

Project description:When irradiated in a transmission electron microscope, plunge-frozen, amorphous water ice specimens accumulate a pattern of static charge that changes dynamically as the specimen is irradiated, and which can deflect the transmitted electrons and blur the resultant micrographs. Here we provide a physical description of this charge accumulation and characterise its dynamic behaviour in the context of low-dose electron cryomicroscopy (cryoEM). We observe the accumulation of positive charge in the primary irradiation area as expected from earlier work. To our surprise, we also observed a build-up of negative charge in nearby unirradiated regions of the specimen. Using a standard carbon support foil containing a pure water ice specimen, we collect a portion of this negative charge in the micrometer sized specimen holes which act as electrostatic lenses. These unusual, diverging micro-lenses are extremely sensitive charge detectors that allow us to directly measure the magnitude and dynamics of charge accumulation and neutralisation that occur during cryoEM imaging. Using these measurements, we find that the build-up of charge on the specimen saturates to a dynamic equilibrium at an electron fluence which is orders of magnitude lower than required for a typical low-dose micrograph. The measurements here will guide the development of optimal imaging conditions for biological specimens and contribute to a complete theory of information loss in electron cryomicroscopy.

| S-EPMC5862658 | biostudies-literature

Electron microscopy: Ultrastable gold substrates for electron cryomicroscopy.

Project description: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 that they cannot be aligned with each other to calculate a three-dimensional density. Much of this movement stems from instabilities in the carbon substrates used to support frozen samples in the microscope. Here we demonstrate a gold specimen support that nearly eliminates substrate motion during irradiation. This increases the subnanometer 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.

| S-EPMC4296556 | biostudies-literature

Cryomicroscopy of radiation sensitive specimens on unmodified graphene sheets: reduction of electron-optical effects of charging.

Project description:Images of radiation-sensitive specimens obtained by electron microscopy suffer a reduction in quality beyond that expected from radiation damage alone due to electron beam-induced charging or movement of the specimen. For biological specimens, charging and movement are most severe when they are suspended in an insulating layer of vitreous ice, which is otherwise optimal for preserving hydrated specimens in a near native state. We image biological specimens, including a single particle protein complex and a lipid-enveloped virus in thin, vitreous ice films over suspended sheets of unmodified graphene. We show that in such preparations, the charging of ice, as assessed by electron-optical perturbation of the imaging beam, is eliminated. We also use the same specimen supports to record high resolution images at liquid nitrogen temperature of monolayer paraffin crystals grown over graphene.

| S-EPMC3778975 | biostudies-literature

Computational Methods for Single-Particle Electron Cryomicroscopy.

Project description:Single-particle electron cryomicroscopy (cryo-EM) is an increasingly popular technique for elucidating the three-dimensional structure of proteins and other biologically significant complexes at near-atomic resolution. It is an imaging method that does not require crystallization and can capture molecules in their native states. In single-particle cryo-EM, the three-dimensional molecular structure needs to be determined from many noisy two-dimensional tomographic projections of individual molecules, whose orientations and positions are unknown. The high level of noise and the unknown pose parameters are two key elements that make reconstruction a challenging computational problem. Even more challenging is the inference of structural variability and flexible motions when the individual molecules being imaged are in different conformational states. This review discusses computational methods for structure determination by single-particle cryo-EM and their guiding principles from statistical inference, machine learning, and signal processing that also play a significant role in many other data science applications.

| S-EPMC8412055 | biostudies-literature

Analyzing RNA polymerase III by electron cryomicroscopy.

Project description:Recent electron cryomicroscopy reconstructions have provided new insights into the overall organization of yeast RNA polymerase (Pol) III, responsible for the synthesis of small, non-translated RNAs. The structure of the free Pol III enzyme at 10 Å resolution provides an accurate framework to better understand its overall architecture and the structural organization and functional role of two Pol III-specific subcomplexes. Cryo-EM structures of elongating Pol III bound to DNA/RNA scaffolds show the rearrangement of the Pol III-specific subcomplexes that enclose incoming DNA. In one reconstruction downstream DNA and newly transcribed RNA can be followed over considerably longer distances as in the crystal structure of elongating Pol II. The Pol III transcription machinery is increasingly recognized as a possible target for cancer therapy. The recent cryo-EM reconstructions contribute to the molecular understanding of Pol III transcription as a prerequisite for targeting its components.

| S-EPMC3256353 | biostudies-literature

Structure of TRPV1 channel revealed by electron cryomicroscopy.

Project description:The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. Here, we report the 3D structure of full-length rat TRPV1 channel expressed in the yeast Saccharomyces cerevisiae and purified by immunoaffinity chromatography. We demonstrate that the recombinant purified TRPV1 channel retains its structural and functional integrity and is suitable for structural analysis. The 19-A structure of TRPV1 determined by using single-particle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N- and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion. The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure.

| S-EPMC2396679 | biostudies-literature

Structure of the mitochondrial ATP synthase by electron cryomicroscopy.

Project description:We have determined the structure of intact ATP synthase from bovine heart mitochondria by electron cryomicroscopy of single particles. Docking of an atomic model of the F1-c10 subcomplex into a major segment of the map has allowed the 32 A resolution density to be interpreted as the F1-ATPase, a central and a peripheral stalk and an FO membrane region that is composed of two domains. One domain of FO corresponds to the ring of c-subunits, and the other probably contains the a-subunit, the transmembrane portion of the b-subunit and the remaining integral membrane proteins of FO. The peripheral stalk wraps around the molecule and connects the apex of F1 to the second domain of FO. The interaction of the peripheral stalk with F1-c10 implies that it binds to a non-catalytic alpha-beta interface in F1 and its inclination where it is not attached to F1 suggests that it has a flexible region that can serve as a stator during both ATP synthesis and ATP hydrolysis.

| S-EPMC291849 | biostudies-literature

Gating machinery of InsP3R channels revealed by electron cryomicroscopy.

Project description:Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca(2+) signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure-function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ∼85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca(2+)-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals.

| S-EPMC4804758 | 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