scholarly journals Advanced Cryo-Electron Microscopy Technology: High Resolution Structure of Macromolecules

2016 ◽  
Vol 46 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Jeong Min Chung ◽  
Hyun Suk Jung
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

2021 ◽  
Author(s):  
Nicole Dimos ◽  
Carl P.O. Helmer ◽  
Andrea M. Chanique ◽  
Markus C. Wahl ◽  
Robert Kourist ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Structure Analysis ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Fast Development ◽  
Pharmaceutical Industries ◽  
Exquisite Specificity ◽  
Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

Cryo-Electron Microscopy of Photosystem II: Towards a High-Resolution Structure

1995 ◽  
pp. 2273-2276 ◽  
Author(s):  
Svetla Stoylova ◽  
Paul McPhie ◽  
Toby D. Flint ◽  
Robert C. Ford ◽  
Andreas Holzenburg
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Photosystem Ii ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Jean-Philippe Demers ◽  
Birgit Habenstein ◽  
Antoine Loquet ◽  
Suresh Kumar Vasa ◽  
Karin Giller ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Solid State ◽  
High Resolution ◽  
Type Iii Secretion ◽  
Solid State Nmr ◽  
Type Iii ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals Comparing the Folds of Prions and Other Pathogenic Amyloids

Pathogens ◽  
2018 ◽  
Vol 7 (2) ◽  
pp. 50 ◽  
Author(s):  
José Flores-Fernández ◽  
Vineet Rathod ◽  
Holger Wille
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Amyloid Β ◽  
Paired Helical Filaments ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Incompatibility System ◽  
High Resolution Structure ◽  
Jakob Disease ◽  
Resolution Structure

Pathogenic amyloids are the main feature of several neurodegenerative disorders, such as Creutzfeldt–Jakob disease, Alzheimer’s disease, and Parkinson’s disease. High resolution structures of tau paired helical filaments (PHFs), amyloid-β(1-42) (Aβ(1-42)) fibrils, and α-synuclein fibrils were recently reported using cryo-electron microscopy. A high-resolution structure for the infectious prion protein, PrPSc, is not yet available due to its insolubility and its propensity to aggregate, but cryo-electron microscopy, X-ray fiber diffraction, and other approaches have defined the overall architecture of PrPSc as a 4-rung β-solenoid. Thus, the structure of PrPSc must have a high similarity to that of the fungal prion HET-s, which is part of the fungal heterokaryon incompatibility system and contains a 2-rung β-solenoid. This review compares the structures of tau PHFs, Aβ(1-42), and α-synuclein fibrils, where the β-strands of each molecule stack on top of each other in a parallel in-register arrangement, with the β-solenoid folds of HET-s and PrPSc.

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.

High-Resolution Cryo-Electron Microscopy of Two-Dimensional Protein Crystals

1990 ◽  
Vol 48 (1) ◽  
pp. 84-85
Author(s):  
F. Zemlin ◽  
E. Beckmann
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Radiation Damage ◽  
Protein Crystals ◽  
Objective Lens ◽  
Two Dimensional ◽  
Cryo Electron Microscopy ◽  
Specimen Temperature ◽  
High Resolution Structure ◽  
Long Time

The task in investigating the structure of crystals is to measure the Fourier coefficients in amplitude and phase. Electron crystallography, i.e. electron diffraction combined with electron-microscopical imaging, is a straigthforward method, because there is a priori no “phase problem”. But radiation damage during electron exposure was for a long time an absolutely unbridgeable chasm on the way to high-resolution structure research of two-dimensional protein crystals by electron microscopy. Now, using cryo-electron microscopy and computer image processing, it has been proven possible to overcome this difficulty and achieve high resolution.Here we present a rough description of the recording procedure. We used a helium-cooled superconducting objective lens with 4.5 K specimen temperature, but the procedure should also be applicable at higher specimen temperatures. The radiation damage is reduced at low specimen temperature, but unfortunately there still remains a remarkable deterioration of the crystal with increasing dose, which means that minimum exposure is advisable.

scholarly journals High-resolution cryo-EM using beam-image shift at 200 keV

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 1179-1187 ◽  
Author(s):  
Jennifer N. Cash ◽  
Sarah Kearns ◽  
Yilai Li ◽  
Michael A. Cianfrocco
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Data Collection ◽  
Single Particle ◽  
Particle Motion ◽  
Data Set ◽  
Cryo Electron Microscopy ◽  
Beam Image ◽  
Shift Data ◽  
Resolution Structure

Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200 keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200 keV using beam-image shift and analyzed. This analysis shows that the instrument beam tilt and particle motion initially limited the resolution to 4.9 Å. After particle polishing and iterative rounds of aberration correction in RELION, a 2.8 Å resolution structure could be obtained. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV.

scholarly journals A case for glycerol as an acceptable additive for single-particle cryoEM samples

2022 ◽  
Vol 78 (1) ◽  
Author(s):  
Benjamin Basanta ◽  
Marscha M. Hirschi ◽  
Danielle A. Grotjahn ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Complete Removal ◽  
High Resolution Imaging ◽  
Rabbit Muscle ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Induced Motion ◽  
Buffer Composition ◽  
Resolution Imaging ◽  
Resolution Structure

Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often involve the minimization or the complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, it is shown that the inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by an ∼2.3 Å resolution reconstruction of mouse apoferritin (∼500 kDa) and an ∼3.3 Å resolution reconstruction of rabbit muscle aldolase (∼160 kDa) in the presence of 20%(v/v) glycerol. While it was found that generating thin ice that is amenable to high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion or an inability to pick particles. Overall, these findings indicate that glycerol should not be discounted as a cryoEM sample-buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.

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