scholarly journals Programming conventional electron microscopes for solving ultrahigh-resolution structures of small and macro-molecules

2019 ◽  
Author(s):  
Heng zhou ◽  
Feng Luo ◽  
Zhipu Luo ◽  
Dan Li ◽  
Cong Liu ◽  
...  
Keyword(s):  
Organic Compounds ◽  
Structure Determination ◽  
Low Cost ◽  
Ccd Camera ◽  
Biological Macromolecules ◽  
Hydrogen Atoms ◽  
Ultrahigh Resolution ◽  
Electron Microscopes ◽  
Camera Synchronization

AbstractMicrocrystal electron diffraction (MicroED) is becoming a powerful tool in determining the crystal structures of biological macromolecules and small organic compounds. However, wide applications of this technique are still limited by the special requirement for radiation-tolerated movie-mode camera and the lacking of automated data collection method. Herein, we develop a stage-camera synchronization scheme to minimize the hardware requirements and enable the use of the conventional electron cryo-microscope with single-frame CCD camera, which ensures not only the acquisition of ultrahigh-resolution diffraction data but also low cost in practice. This method renders the structure determination of both peptide and small organic compounds at ultrahigh resolution up to ~0.60 Å with unambiguous assignment of nearly all hydrogen atoms. The present work provides a widely applicable solution for routine structure determination of MicroED, and demonstrates the capability of the low-end 120kV microscope with a CCD camera in solving ultra-high resolution structures of both organic compound and biological macromolecules.

scholarly journals Biofilm Localization in the Vertical Wall of Shaking 96-Well Plates

Scientifica ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Luciana C. Gomes ◽  
Joana M. R. Moreira ◽  
Manuel Simões ◽  
Luís F. Melo ◽  
Filipe J. Mergulhão
Keyword(s):  
Low Cost ◽  
Vertical Wall ◽  
Spatial Location ◽  
Strain Rates ◽  
Microtiter Plates ◽  
Equipment Availability ◽  
Cell Accumulation ◽  
Electron Microscopes ◽  
Air Liquid Interface

Microtiter plates with 96 wells are being increasingly used for biofilm studies due to their high throughput, low cost, easy handling, and easy application of several analytical methods to evaluate different biofilm parameters. These methods provide bulk information about the biofilm formed in each well but lack in detail, namely, regarding the spatial location of the biofilms. This location can be obtained by microscopy observation using optical and electron microscopes, but these techniques have lower throughput and higher cost and are subjected to equipment availability. This work describes a differential crystal violet (CV) staining method that enabled the determination of the spatial location ofEscherichia colibiofilms formed in the vertical wall of shaking 96-well plates. It was shown that the biofilms were unevenly distributed on the wall with denser cell accumulation near the air-liquid interface. The results were corroborated by scanning electron microscopy and a correlation was found between biofilm accumulation and the wall shear strain rates determined by computational fluid dynamics. The developed method is quicker and less expensive and has a higher throughput than the existing methods available for spatial location of biofilms in microtiter plates.

scholarly journals Breaking the next Cryo-EM resolution barrier – Atomic resolution determination of proteins!

2020 ◽  
Author(s):  
Ka Man Yip ◽  
Niels Fischer ◽  
Elham Paknia ◽  
Ashwin Chari ◽  
Holger Stark
Keyword(s):  
Protein Function ◽  
Model Building ◽  
Technological Development ◽  
Drug Binding ◽  
Single Atom ◽  
Biological Macromolecules ◽  
Direct Visualization ◽  
Hydrogen Atoms ◽  
Structure Based Drug Design ◽  
Electron Microscopes

SummarySingle particle cryo-EM is a powerful method to solve the three-dimensional structures of biological macromolecules. The technological development of electron microscopes, detectors, automated procedures in combination with user friendly image processing software and ever-increasing computational power have made cryo-EM a successful and largely expanding technology over the last decade. At resolutions better than 4 Å, atomic model building starts becoming possible but the direct visualization of true atomic positions in protein structure determination requires significantly higher (< 1.5 Å) resolution, which so far could not be attained by cryo-EM. The direct visualization of atom positions is essential for understanding protein-catalyzed chemical reaction mechanisms and to study drug-binding and -interference with protein function. Here we report a 1.25 Å resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope providing unprecedented structural details. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution 1). For the first time in cryo-EM we can visualize individual atoms in a protein, see density for hydrogen atoms and single atom chemical modifications. Beyond the nominal improvement in resolution we can also show a significant improvement in quality of the cryo-EM density map which is highly relevant for using cryo-EM in structure-based drug design.

Orientation Analysis by Computer-Aided Diffraction Pattern Matching in The TEM

1997 ◽  
Vol 3 (S2) ◽  
pp. 1017-1018
Author(s):  
L. E. Thomas ◽  
R. C. Hugo ◽  
J. T. Stanley
Keyword(s):  
Ccd Camera ◽  
Precise Determination ◽  
Diffraction Spot ◽  
Phase Identification ◽  
Desktop Computer ◽  
Computer Aided ◽  
Viewing Screen ◽  
Electron Microscopes ◽  
Diffraction Patterns

Electron diffraction patterns taken in transmission electron microscopes are widely used for phase identification and orientation determination of crystallites as small as hundreds or even tens of nanometers in size. The analyses typically require rather tedious measurement of the patterns, and matching of calculated d-spacings and angles with those of known phases. Recently, the analysis of these patterns has been facilitated by powerful desktop computer programs that use digitally captured images for on-line measurement and simulation matching of" the diffraction spot and Kikuchi line patterns. This presentation will illustrate an application of computer-aided pattern simulation and matching for precise determination of crystal orientations.Two experimental TV camera arrangements were used to record diffraction patterns in a TEM. These included a cooled-CCD camera located on the electron optical axis below the microscope viewing chamber, and a simple TV-rate CCD that recorded directly from the inclined fluorescent viewing screen of the microscope.

Structure determination of organic compounds by ?electron pyrolysis?

1977 ◽  
Vol 286 (5) ◽  
pp. 355-360 ◽  
Author(s):  
G. Ege ◽  
K. Ge�ner ◽  
H. Schildknecht
Keyword(s):  
Organic Compounds ◽  
Structure Determination

scholarly journals High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

2018 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Drug Target ◽  
Phase Plate ◽  
Biological Macromolecules ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
High Resolution Structure ◽  
Resolution Structure

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

Structure Determination of Organic Compounds

2020 ◽  
Author(s):  
Ernö Pretsch ◽  
Philippe Bühlmann ◽  
Martin Badertscher
Keyword(s):  
Organic Compounds ◽  
Structure Determination
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