scholarly journals Sub-2 Å Resolution Structure Determination Using Single-Particle Cryo-EM at 200 keV

2019 ◽  
Author(s):  
Mengyu Wu ◽  
Gabriel C. Lander ◽  
Mark A. Herzik
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Single Particle ◽  
Structure Based Drug Design ◽  
Transmission Electron ◽  
Electron Microscopes ◽  
Ordered Water ◽  
Current Paradigm ◽  
Resolution Single

AbstractAlthough 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 keV for high-resolution single-particle cryo-EM and applications such as structure-based drug design.

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.

scholarly journals Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV

2017 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
Single Particle ◽  
Water Molecules ◽  
Macromolecular Complexes ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Electron Microscopes ◽  
Ordered Water ◽  
Better Than

AbstractTechnical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 Å resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 Å resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.

Advances in image simulation for high-resolution TEM

1995 ◽  
Vol 53 ◽  
pp. 38-39
Author(s):  
Michael A. O'Keefe
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Incident Beam ◽  
Hrtem Image ◽  
Image Simulation ◽  
Lattice Image ◽  
Transmission Electron ◽  
High Resolution Tem ◽  
Projection Approximation

The original high-resolution transmission electron microscope (HRTEM) image simulation program was written as a tool to confirm interpretation of HRTEM images of niobium oxides. Thorough testing on known structures showed that image simulation could reliably duplicate the imaging process occurring in the HRTEM, and could thus be confidently used to interpret images of unknown structures. Mainstream application of image simulation to routine structure determination by HRTEM was ushered in by the establishment of the wide applicability of the SHRLI (simulated high-resolution lattice image) programs. Structure determination of the mineral takéuchiite by HRTEM and image simulation was the first such determination accepted by the KJCr without x-ray data. Of course, once the reliability of image simulation had been established, it was realized that the technique could be put to work for applications other than structure determination. Early on, simulations were used to explore various HRTEM imaging parameters, including specimen ionicity, validity of the projection approximation, and the resolutionlimiting effects of incident-beam convergence. Since the inception of HRTEM image simulation, its range of uses has continued to expand, and so has the number of programs available; distribution of the SHRLI code spawned improved versions as well as some new programs.

scholarly journals Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution

2021 ◽  
Author(s):  
Ivan Lazic ◽  
Maarten Wirix ◽  
Max Leo Leidl ◽  
Felix de Haas ◽  
Maximilian Beckers ◽  
...  
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Single Particle ◽  
3D Structure ◽  
Atomic Resolution ◽  
Biological Macromolecules ◽  
Data Set ◽  
Helical Reconstruction ◽  
Transmission Electron ◽  
Scanning Transmission

Electron cryo-microscopy (cryo-EM) is becoming one of the routine tools for structure determination of biological macromolecules. Commonly, molecular images are obtained by conventional transmission electron microcopy (CTEM) using underfocus and subsequently computationally combined into a high-resolution 3D structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to the cryo-EM test specimen of tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination at 3.5 Angstrom resolution using single-particle based helical reconstruction. A series of cryo-EM TMV maps was resolved at near-atomic resolution taken at different convergence semi-angle (CSA) beams and share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. The associated map B-factors from iDPC-STEM match those obtained by CTEM recordings using 2nd generation direct electron detection devices. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.

High-resolution microscopy of twin boundaries in gold

1987 ◽  
Vol 45 ◽  
pp. 260-261
Author(s):  
William Krakow ◽  
David A. Smith
Keyword(s):  
High Resolution ◽  
Specimen Preparation ◽  
Gold Films ◽  
Boundary Area ◽  
Transmission Electron ◽  
Recent Developments ◽  
Tilt Boundaries ◽  
High Resolution Microscopy ◽  
Twist Boundaries

Recent developments in specimen preparation, imaging and image analysis together permit the experimental determination of the atomic structure of certain, simple grain boundaries in metals such as gold. Single crystal, ∼125Å thick, (110) oriented gold films are vapor deposited onto ∼3000Å of epitaxial silver on (110) oriented cut and polished rock salt substrates. Bicrystal gold films are then made by first removing the silver coated substrate and placing in contact two suitably misoriented pieces of the gold film on a gold grid. Controlled heating in a hot stage first produces twist boundaries which then migrate, so reducing the grain boundary area, to give mixed boundaries and finally tilt boundaries perpendicular to the foil. These specimens are well suited to investigation by high resolution transmission electron microscopy.

Comparison of ultra high resolution immersion lens CFESEM and TEM for imaging of semiconductor devices

1990 ◽  
Vol 48 (4) ◽  
pp. 622-623
Author(s):  
Terrence Reilly ◽  
Al Pelillo ◽  
Barbara Miner
Keyword(s):  
High Resolution ◽  
Sample Preparation ◽  
Cross Sections ◽  
Semiconductor Devices ◽  
Ion Milling ◽  
Observation Area ◽  
Transmission Electron ◽  
Milling Machines ◽  
Resolution Imaging ◽  
Electron Microscopes

The use of transmission electron microscopes (TEM) has proven to be very valuable in the observation of semiconductor devices. The need for high resolution imaging becomes more important as the devices become smaller and more complex. However, the sample preparation for TEM observation of semiconductor devices have generally proven to be complex and time consuming. The use of ion milling machines usually require a certain degree of expertise and allow a very limited viewing area. Recently, the use of an ultra high resolution "immersion lens" cold cathode field emission scanning electron microscope (CFESEM) has proven to be very useful in the observation of semiconductor devices. Particularly at low accelerating voltages where compositional contrast is increased. The Hitachi S-900 has provided comparable resolution to a 300kV TEM on semiconductor cross sections. Using the CFESEM to supplement work currently being done with high voltage TEMs provides many advantages: sample preparation time is greatly reduced and the observation area has also been increased to 7mm. The larger viewing area provides the operator a much greater area to search for a particular feature of interest. More samples can be imaged on the CFESEM, leaving the TEM for analyses requiring diffraction work and/or detecting the nature of the crystallinity.

A Structural Model for a Quasi-Crystalline Material Derived from TEM

1990 ◽  
Vol 48 (1) ◽  
pp. 48-49
Author(s):  
Jan-Olov Bovin ◽  
Osamu Terasaki ◽  
Jan-Olle Malm ◽  
Sven Lidin ◽  
Sten Andersson
Keyword(s):  
High Resolution ◽  
Electron Diffraction ◽  
Structural Model ◽  
Image Intensifier ◽  
Crystalline Materials ◽  
Icosahedral Phases ◽  
Transmission Electron ◽  
Electron Microscopes ◽  
Diffraction Patterns ◽  
Quasi Crystals

High resolution transmission electron microscopy (HRTEM) is playing an important role in identifying the new icosahedral phases. The selected area diffraction patterns of quasi crystals, recorded with an aperture of the radius of many thousands of Ångströms, consist of dense arrays of well defined sharp spots with five fold dilatation symmetry which makes the interpretation of the diffraction process and the resulting images different from those invoked for usual crystals. The atomic structure of the quasi crystals is not established even if several models are proposed. The correct structure model must of course explain the electron diffraction patterns with 5-, 3- and 2-fold symmetry for the phases but it is also important that the HRTEM images of the alloys match the computer simulated images from the model. We have studied quasi crystals of the alloy Al65Cu20Fe15. The electron microscopes used to obtain high resolution electro micrographs and electron diffraction patterns (EDP) were a (S)TEM JEM-2000FX equipped with EDS and PEELS showing a structural resolution of 2.7 Å and a IVEM JEM-4000EX with a UHP40 high resolution pole piece operated at 400 kV and with a structural resolution of 1.6 Å. This microscope is used with a Gatan 622 TV system with an image intensifier, coupled to a YAG screen. It was found that the crystals of the quasi crystalline materials here investigated were more sensitive to beam damage using 400 kV as electron accelerating voltage than when using 200 kV. Low dose techniques were therefore applied to avoid damage of the structure.

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