Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions.

ABSTRACT: The Ewald sphere effect is generally neglected when using the Central Projection Theorem for cryo electron microscopy single-particle reconstructions. This can reduce the resolution of a reconstruction. Here we estimate the attainable resolution and report a "block-based" reconstruction method for extending the resolution limit. We find the Ewald sphere effect limits the resolution of large objects, especially large viruses. After processing two real datasets of large viruses, we show that our procedure can extend the resolution for both datasets and can accommodate the flexibility associated with large protein complexes.

SUBMITTER: Zhu D

PROVIDER: S-EPMC5908801 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions.

Zhu Dongjie D Wang Xiangxi X Fang Qianglin Q Van Etten James L JL Rossmann Michael G MG Rao Zihe Z Zhang Xinzheng X

Nature communications 20180419 1

The Ewald sphere effect is generally neglected when using the Central Projection Theorem for cryo electron microscopy single-particle reconstructions. This can reduce the resolution of a reconstruction. Here we estimate the attainable resolution and report a "block-based" reconstruction method for extending the resolution limit. We find the Ewald sphere effect limits the resolution of large objects, especially large viruses. After processing two real datasets of large viruses, we show that our p ...[more]

PMID: 29674632

Similar Datasets

Single-particle cryo-EM at atomic resolution.

Project description:The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

| S-EPMC7611073 | biostudies-literature

High-resolution cryo-EM reconstructions in the presence of substantial aberrations.

Project description:Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula quantifying information loss owing to their presence is inferred that explains why Fourier-shell coefficient-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. The analysis is validated with reference-based aberration refinement for two cryo-EM SPR data sets acquired with a 200?kV microscope in the presence of coma exceeding 40?µm, and 2.3 and 2.7?Å reconstructions for 144 and 173?kDa particles, respectively, were obtained. The results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher order aberrations, and address inconsistent guidelines regarding the level of aberrations that is acceptable in cryo-EM SPR experiments.

| S-EPMC7201289 | biostudies-literature

Single Particle Cryo-EM Reconstructions of NCP-CHD4 complexes

Project description: Not available

| EMPIAR-10411 | biostudies-other

From Ewald sphere to Ewald shell in nonlinear optics.

Project description:Ewald sphere is a simple vector scheme to depict the X-ray Bragg diffraction in a crystal. A similar method, known as the nonlinear Ewald sphere, was employed to illustrate optical frequency conversion processes. We extend the nonlinear Ewald sphere to the Ewald shell construction. With the Ewald shell, a variety of quasi-phase-matching (QPM) effects, such as the collective envelope effect associated with multiple QPM resonances, the enhanced second- harmonic generation due to multiple reciprocal vectors etc., are suggested theoretically and verified experimentally. By rotating the nonlinear photonic crystal sample, the dynamic evolution of these QPM effects has also been observed, which agreed well with the Ewald shell model.

| S-EPMC4937392 | biostudies-literature

Cryo-EM model validation using independent map reconstructions.

Project description:An increasing number of cryo-electron microscopy (cryo-EM) density maps are being generated with suitable resolution to trace the protein backbone and guide sidechain placement. Generating and evaluating atomic models based on such maps would be greatly facilitated by independent validation metrics for assessing the fit of the models to the data. We describe such a metric based on the fit of atomic models with independent test maps from single particle reconstructions not used in model refinement. The metric provides a means to determine the proper balance between the fit to the density and model energy and stereochemistry during refinement, and is likely to be useful in determining values of model building and refinement metaparameters quite generally.

| S-EPMC3690725 | biostudies-literature

Retrieving high-resolution information from disordered 2D crystals by single-particle cryo-EM.

Project description:Electron crystallography can reveal the structure of membrane proteins within 2D crystals under close-to-native conditions. High-resolution structural information can only be reached if crystals are perfectly flat and highly ordered. In practice, such crystals are difficult to obtain. Available image unbending algorithms correct for disorder, but only perform well on images of non-tilted, flat crystals, while out-of-plane distortions are not addressed. Here, we present an approach that employs single-particle refinement procedures to locally unbend crystals in 3D. With this method, density maps of the MloK1 potassium channel with a resolution of 4 Å were obtained from images of 2D crystals that do not diffract beyond 10 Å. Furthermore, 3D classification allowed multiple structures to be resolved, revealing a series of MloK1 conformations within a single 2D crystal. This conformational heterogeneity explains the poor diffraction observed and is related to channel function. The approach is implemented in the FOCUS package.

| S-EPMC6461647 | biostudies-literature

Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV.

Project description:Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7 Å resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 kV electrons for high-resolution single-particle cryo-EM and applications such as structure-based drug design.

| S-EPMC7337053 | biostudies-literature

High-resolution single-particle cryo-EM of samples vitrified in boiling nitro-gen.

Project description:Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures. These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. However, ethane and propane are flammable, they must be liquified in liquid nitro-gen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitro-gen for storage, complicating workflows and increasing the chance of sample damage during handling. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only ∼250 000 K s-1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitro-gen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect. Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitro-gen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitro-gen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.

| S-EPMC8562666 | biostudies-literature

Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution.

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.

| S-EPMC6546690 | biostudies-literature

Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM.

Project description:Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4?Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1?Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.

| S-EPMC7381653 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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