scholarly journals MicroED with the Falcon III direct electron detector

2019 ◽  
Author(s):  
Johan Hattne ◽  
Michael W. Martynowycz ◽  
Tamir Gonen
Keyword(s):  
High Energy ◽  
Proteinase K ◽  
Model Quality ◽  
Electron Detector ◽  
Transmission Electron ◽  
High Energy Electrons ◽  
High Resolution Structure ◽  
Dose Radiation ◽  
Resolution Structure ◽  
Nanosized Crystals

AbstractMicrocrystal electron diffraction (MicroED) combines crystallography and electron cryomicroscopy (cryo-EM) into a method that can be used for high-resolution structure determination. In MicroED nanosized crystals, often intractable by other techniques, are probed by high-energy electrons in a transmission electron microscope and the diffracted signal is recorded on an electron detector. Since only a small number of different detectors have been used for MicroED measurements in the past, their impact on data quality has not been investigated. Here we evaluate two different cameras using crystals of the well-characterized serine protease proteinase K. Compared to previously used equipment, the Falcon III direct electron detector and the CMOS-based CetaD camera can collect complete datasets both faster and using lower total exposure. As an effect of the lower dose, radiation damage is reduced, which is confirmed in both real and reciprocal space. The increased speed and lower exposure requirements have implications on model quality and the prospects for further automation of MicroED.

scholarly journals MicroED with the Falcon III direct electron detector

IUCrJ ◽  
2019 ◽  
Vol 6 (5) ◽  
pp. 921-926 ◽  
Author(s):  
Johan Hattne ◽  
Michael W. Martynowycz ◽  
Pawel A. Penczek ◽  
Tamir Gonen
Keyword(s):  
High Energy ◽  
Proteinase K ◽  
Data Sets ◽  
Electron Detector ◽  
Density Maps ◽  
Transmission Electron ◽  
High Energy Electrons ◽  
High Resolution Structure ◽  
Resolution Structure ◽  
Nanosized Crystals

Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.

scholarly journals Devitrification reduces beam-induced movement in cryo-EM

IUCrJ ◽  
2021 ◽  
Vol 8 (2) ◽  
pp. 186-194
Author(s):  
Jan-Philip Wieferig ◽  
Deryck J. Mills ◽  
Werner Kühlbrandt
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
High Energy ◽  
Full Potential ◽  
High Energy Electrons ◽  
High Resolution Structure ◽  
Image Blurring ◽  
Particle Images ◽  
Resolution Structure

As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent problems has been beam-induced movement, which occurs when the specimen is irradiated with high-energy electrons. Beam-induced movement results in image blurring and loss of high-resolution information. It is particularly severe for biological samples in unsupported thin films of vitreous water. By controlled devitrification of conventionally plunge-frozen samples, the suspended film of vitrified water was converted into cubic ice, a polycrystalline, mechanically stable solid. It is shown that compared with vitrified samples, devitrification reduces beam-induced movement in the first 5 e Å−2 of an exposure by a factor of ∼4, substantially enhancing the contribution of the initial, minimally damaged frames to a structure. A 3D apoferritin map reconstructed from the first frames of 20 000 particle images of devitrified samples resolved undamaged side chains. Devitrification of frozen-hydrated specimens helps to overcome beam-induced specimen motion in single-particle cryo-EM, as a further step towards realizing the full potential of cryo-EM for high-resolution 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.

scholarly journals High-resolution structure of proteinase K cocrystallized with digalacturonic acid

2009 ◽  
Vol 65 (3) ◽  
pp. 192-198 ◽  
Author(s):  
Steven B. Larson ◽  
John S. Day ◽  
Chieugiang Nguyen ◽  
Robert Cudney ◽  
Alexander McPherson
Keyword(s):  
High Resolution ◽  
Proteinase K ◽  
High Resolution Structure ◽  
Resolution Structure

A Novel Diode Detector in a Scanning Transmission Microscope

1972 ◽  
Vol 30 ◽  
pp. 442-443
Author(s):  
C. S. Kim ◽  
T. E. Everhart
Keyword(s):  
High Energy ◽  
Scanning Transmission Electron Microscope ◽  
Semiconductor Diode ◽  
Objective Lens ◽  
Contrast Effects ◽  
Thin Sample ◽  
Transmission Electron ◽  
High Energy Electrons ◽  
Scanning Transmission ◽  
Pass Through

High-resolution in a scanning transmission electron microscope can be obtained using a condenser-objective lens. A suitable semiconductor diode is an efficient detector of high-energy electrons; an annular detector allows unscattered primary electrons or inelastically scattered electrons to pass through the hole, while elastically scattered electrons strike the diode, and are detected.Electrons passing through a thin sample may be elastically scattered through angles of many tens of milliradians, inelastically scattered with angular deflections of ∼ 1 mr, or not scattered at all. The inelastically scattered electrons do not depart significantly from the unscattered beam. Since the beam convergence angle at the sample is typically a few milliradians, the elastically scattered electrons can be collected using a detector with a hole positioned at the beam axis to allow the inelastically scattered electrons and the unscattered electrons to pass through. These electrons can be separated with an electron spectrometer to provide important contrast effects.

Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

2020 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Tamir Gonen
Keyword(s):  
High Resolution ◽  
Electron Diffraction ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Proteinase K ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Resolution Structure ◽  
Efficient Uptake ◽  
Resolution Structure

AbstractA method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoEM vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of 200nm using a focused ion-beam, and microcrystal electron diffraction (MicroED) data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Compared to much larger crystals investigated by X-ray crystallography, both the number of ligands bound and their occupancy was higher in MicroED. These results indicate that soaking ligands into microcrystals in this way results in a more efficient uptake than in larger crystals that are typically used in drug discovery pipelines by X-ray crystallography.

scholarly journals Investigation of Nanoelectrodes by Transmission Electron Microscopy

2001 ◽  
Vol 676 ◽  
Author(s):  
M. S. Kabir ◽  
S. H. Magnus Persson ◽  
Yimin Yao ◽  
Jean Phillippe Bourgoin ◽  
Serge Palacin
Keyword(s):  
Room Temperature ◽  
High Energy ◽  
Image Features ◽  
Electrical Characteristics ◽  
Conjugated Molecules ◽  
Atomic Force ◽  
Transmission Electron ◽  
High Energy Electrons ◽  
Electron Microscopes ◽  
Scanning Electron Microscopes

ABSTRACTElectrodes for making connections to single molecules and clusters must have separations smaller than 10 nm. They are therefore difficult or impossible to image with atomic force microscopes (AFM) or Scanning Electron Microscopes (SEM). We have fabricated nanoelelectrodes by different methods to contacts nanoclusters and conjugated molecules and investigated their properties in transmission electron microscope (TEM) and their electrical characteristics at room temperature and at 4.2K. The electrodes are made on SiN4 membranes, which is transparent to high energy electrons and which make it possible to image features of a few nanometers in TEM.

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