Conquer by cryo-EM without physically dividing

2021 ◽  
Author(s):  
Gabriel C. Lander ◽  
Robert M. Glaeser
Keyword(s):  
Particle Size ◽  
High Resolution ◽  
Single Particle ◽  
Region Of Interest ◽  
Divide And Conquer ◽  
Particle Sizes ◽  
X Ray ◽  
X Ray Crystallography ◽  
Substantial Progress ◽  
Made In

This mini-review provides an update regarding the substantial progress that has been made in using single-particle cryo-EM to obtain high-resolution structures for proteins and other macromolecules whose particle sizes are smaller than 100 kDa. We point out that establishing the limits of what can be accomplished, both in terms of particle size and attainable resolution, serves as a guide for what might be expected when attempting to improve the resolution of small flexible portions of a larger structure using focused refinement approaches. These approaches, which involve computationally ignoring all but a specific, targeted region of interest on the macromolecules, is known as ‘masking and refining,' and it thus is the computational equivalent of the ‘divide and conquer' approach that has been used so successfully in X-ray crystallography. The benefit of masked refinement, however, is that one is able to determine structures in their native architectural context, without physically separating them from the biological connections that they require for their function. This mini-review also compares where experimental achievements currently stand relative to various theoretical estimates for the smallest particle size that can be successfully reconstructed to high resolution. Since it is clear that a substantial gap still remains between the two, we briefly recap the areas in which further improvement seems possible, both in equipment and in methods.

scholarly journals On the explicit use of experimental images in high resolution cryo-EM refinement

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 665
Author(s):  
Jacqueline Cherfils ◽  
Jorge Navaza
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Single Particle ◽  
Atomic Resolution ◽  
Full Potential ◽  
X Ray ◽  
X Ray Crystallography ◽  
Atomic Models

Single particle cryogenic electron microscopy (cryo-EM) is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography. As the technique continues to improve, it is important to assess and exploit its full potential to produce the most possible reliable atomic models. Here we propose to use the experimental images as the data for refinement and validation, instead of the reconstructed maps as currently used. This procedure, which is in spirit quite similar to that used in X-ray crystallography where the data include experimental phases, should contribute to improve the quality of the cryo-EM atomic models.

scholarly journals On the explicit use of experimental images in high resolution cryo-EM refinement

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 665
Author(s):  
Jacqueline Cherfils ◽  
Jorge Navaza
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Membrane Proteins ◽  
Structural Biology ◽  
Single Particle ◽  
Atomic Resolution ◽  
Full Potential ◽  
X Ray ◽  
X Ray Crystallography

Single particle cryogenic electron microscopy (cryo-EM) is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography. As the technique continues to improve, it is important to assess and exploit its full potential to produce the most possible reliable atomic models. Here we propose to use the experimental images as the data for refinement and validation, instead of the reconstructed maps as currently used. This procedure, which is in spirit quite similar to that used in X-ray crystallography where the data include experimental phases, should contribute to improve the quality of the models.

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

scholarly journals ZnWO4 Nanoparticle Scintillators for High Resolution X-ray Imaging

Nanomaterials ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1721
Author(s):  
Heon Yong Jeong ◽  
Hyung San Lim ◽  
Ju Hyuk Lee ◽  
Jun Heo ◽  
Hyun Nam Kim ◽  
...  
Keyword(s):  
Particle Size ◽  
Zinc Oxide ◽  
High Resolution ◽  
Tungsten Oxide ◽  
Spatial Resolution ◽  
High Spatial Resolution ◽  
X Ray ◽  
X Ray Imaging ◽  
Average Size ◽  
And Tungsten

The effect of scintillator particle size on high-resolution X-ray imaging was studied using zinc tungstate (ZnWO4) particles. The ZnWO4 particles were fabricated through a solid-state reaction between zinc oxide and tungsten oxide at various temperatures, producing particles with average sizes of 176.4 nm, 626.7 nm, and 2.127 μm; the zinc oxide and tungsten oxide were created using anodization. The spatial resolutions of high-resolution X-ray images, obtained from utilizing the fabricated particles, were determined: particles with the average size of 176.4 nm produced the highest spatial resolution. The results demonstrate that high spatial resolution can be obtained from ZnWO4 nanoparticle scintillators that minimize optical diffusion by having a particle size that is smaller than the emission wavelength.

Three-dimensional crystallization of membrane proteins

1988 ◽  
Vol 21 (4) ◽  
pp. 429-477 ◽  
Author(s):  
W. Kühlbrandt
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Membrane Protein Complexes ◽  
Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

Use of Ceramic Waste for the Manufacture of Sintered Parts

2020 ◽  
Vol 1012 ◽  
pp. 233-238
Author(s):  
Vanessa Moura de Souza ◽  
Vinícius Martins ◽  
Rejane Maria Candiota Tubino
Keyword(s):  
Particle Size ◽  
Selection Process ◽  
Compaction Pressure ◽  
Raw Material ◽  
Powder Technology ◽  
Particle Sizes ◽  
Compression Pressure ◽  
X Ray ◽  
Ceramic Waste ◽  
Quality Process

This paper evaluated the use of the pitcher, a ceramic waste obtained through the quality process of a sanitary ware industry, in the development of a material for usage in the manufacture of sintered parts. The pitcher was obtained through powder technology and is composed, according to the chemical analysis obtained by X-ray fluorescence spectrometry, of clayey minerals (clay and kaolin), quartz, and feldspar, which may include ceramic rocks such as granite, pegmatite and phyllite; that is, it has proved to be a potential raw material due to the minerals that can still be reused. The pitcher passed through a granulometry-based selection process, sieving about 20kg using the following sieve sequence: 18 MESH, 25 MESH, 30 MESH, 120 MESH and 400 MESH; with around 70% of the residue being retained in the sieves of 120 and 400 MESH, which were selected to be used in the evaluation. The samples were compacted in a manual press with different pressures, between 300 and 1000 kgf, and after were sintered at a temperature of 1100oC in a resistive furnace. To characterize the material, the apparent and green density, as well as the compressibility curve, were determined to identify the best compression pressure. The microstructure of the test specimen and the pitcher homogeneity were evaluated using Scanning Electron Microscopy (SEM). Both particle sizes presented the typical compressibility curve, in which the density increases with increasing compaction pressure, while the curve slope decreases with increasing pressure. The density increase with the increasing compaction pressure indicates a good densification for the temperatures, independent of the sample granulometry. The sintering porosity decreased proportionally to the particle size in the sintered samples. The analysis showed that the particle size of 400 MESH sintered at 1100oC obtained more porous surfaces, thus indicating a promising future for the manufacture of parts using powder technology, especially for the development of filters.

scholarly journals Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1211-C1211
Author(s):  
Joseph Ng ◽  
Ronny Hughes ◽  
Michelle Morris ◽  
Leighton Coates ◽  
Matthew Blakeley ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Site ◽  
Inorganic Pyrophosphatase ◽  
Inorganic Pyrophosphate ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Crystallographic Structures ◽  
Hydrolysis Of ◽  
Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

scholarly journals Theoretical and Experimental Evaluation of Particle size Effect of Iron , Cobalt ,and Nickel Powders Suspended in Al Dura oil on XRF Intensities

2007 ◽  
Vol 4 (3) ◽  
pp. 475-481
Author(s):  
Baghdad Science Journal
Keyword(s):  
Particle Size ◽  
Particle Size Distribution ◽  
Laser System ◽  
Particle Size Effect ◽  
Particle Sizes ◽  
Iron Cobalt ◽  
X Ray ◽  
Nickel Powders ◽  
Good Agreement ◽  
Cobalt And Nickel

Iron , Cobalt , and Nickel powders with different particle sizes were subjected to sieving and He-Ne laser system to determine the particle size . 1wt% from each powders was blended carefully with 99wt% from Iraqi oil . Microscopic examination were carried for all samples to reveal the particle size distribution . A Siemens type SRS sequential wavelength dispersive(WDS) X-ray spectrometer was used to analyze all samples , and the XRF intensity were determined experimentally and theoretically for all suspended samples , Good agreement between theoretical and experimental results were found .

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