scholarly journals A resolution record for cryoEM

2021 ◽  
Vol 10 ◽  
Author(s):  
Jonathan Ashmore ◽  
Bridget Carragher ◽  
Peter B Rosenthal ◽  
William Weis
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Structure Determination ◽  
Test Specimen ◽  
Structural Information ◽  
Atomic Resolution ◽  
Fast Growing ◽  
Cryo Electron Microscopy ◽  
New Instrument ◽  
Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

scholarly journals Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

2018 ◽  
Author(s):  
Diego Gauto ◽  
Leandro Estrozi ◽  
Charles Schwieters ◽  
Gregory Effantin ◽  
Pavel Macek ◽  
...  
Keyword(s):  
Structure Determination ◽  
Protein Function ◽  
Structural Information ◽  
Magic Angle Spinning ◽  
Atomic Resolution ◽  
Magic Angle ◽  
Nmr Structure Determination ◽  
Structure Information ◽  
Resolution Structure

Atomic-resolution structure determination is the key requirement for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enabled determination of the high-resolution structure of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Angstrom by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and specifically labelled methyl groups, and a 4.1 Angstrom resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution (up to 8 Angstrom) cryo-EM data are available, thus paving avenues for the structure determination of challenging biological assemblies.

scholarly journals Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age

2019 ◽  
Vol 75 (1) ◽  
pp. 3-11 ◽  
Author(s):  
Alok K. Mitra
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particles ◽  
Cryo Electron Microscopy ◽  
Electron Microscopes ◽  
Resolution Single ◽  
Resolution Structure

Structural biology is going through a revolution as a result of transformational advances in the field of cryo-electron microscopy (cryo-EM) driven by the development of direct electron detectors and ultrastable electron microscopes. High-resolution cryo-EM images of isolated biomolecules (single particles) suspended in a thin layer of vitrified buffer are subjected to powerful image-processing algorithms, enabling near-atomic resolution structures to be determined in unprecedented numbers. Prior to these advances, electron crystallography of two-dimensional crystals and helical assemblies of proteins had established the feasibility of atomic resolution structure determination using cryo-EM. Atomic resolution single-particle analysis, without the need for crystals, now promises to resolve problems in structural biology that were intractable just a few years ago.

scholarly journals Resolving Individual-Atom of Protein Complex using Commonly Available 300-kV Cryo-electron Microscopes

2020 ◽  
Author(s):  
Kaiming Zhang ◽  
Grigore D. Pintilie ◽  
Shanshan Li ◽  
Michael F. Schmid ◽  
Wah Chiu
Keyword(s):  
Structure Determination ◽  
Single Particle ◽  
Heavy Atom ◽  
Atomic Resolution ◽  
Water Molecules ◽  
Hydrogen Atoms ◽  
Cryo Electron Microscopy ◽  
Resolution Model ◽  
Electron Microscopes ◽  
Resolution Structure

AbstractBreakthroughs in single-particle cryo-electron microscopy (cryo-EM) technology have made near-atomic resolution structure determination possible. Here, we report a ∼1.35-Å structure of apoferritin reconstructed from images recorded on a Gatan K3 or a Thermo Fisher Falcon 4 detector in a commonly available 300-kV Titan Krios microscope (G3i) equipped with or without a Gatan post-column energy filter. Our results demonstrate that the atomic-resolution structure determination can be achieved by single-particle cryo-EM with a fraction of a day of automated data collection. These structures resolve unambiguously each heavy atom (C, N, O, and S) in the amino acid side chains with an indication of hydrogen atoms’ presence and position, as well as the unambiguous existence of multiple rotameric configurations for some residues. We also develop a statistical and chemical based protocol to assess the positions of the water molecules directly from the cryo-EM map. In addition, we have introduced a B’ factor equivalent to the conventional B factor traditionally used by crystallography to annotate the atomic resolution model for determined structures. Our findings will be of immense interest among protein and medicinal scientists engaging in both basic and translational research.

scholarly journals CryoEM structure of the antibacterial target PBP1b at 3.3 Å resolution

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nathanael A. Caveney ◽  
Sean D. Workman ◽  
Rui Yan ◽  
Claire E. Atkinson ◽  
Zhiheng Yu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Acid Anhydride ◽  
Atomic Resolution ◽  
Inherent Difficulty ◽  
Penicillin Binding Proteins ◽  
Antibiotic Development ◽  
Class A ◽  
Membrane Mimetic ◽  
Antibacterial Target ◽  
Resolution Structure

AbstractThe pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of β-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.

scholarly journals In situ structure determination using single particle cryo-electron microscopy images

2020 ◽  
Author(s):  
Jing Cheng ◽  
Bufan Li ◽  
Long Si ◽  
Xinzheng Zhang
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Structure Refinement ◽  
Target Function ◽  
Target Protein ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Tilt Series

AbstractCryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

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.

scholarly journals Structure of the Infective Getah Virus at 2.8 Å-resolution Determined by Cryo-electron Microscopy

2021 ◽  
Author(s):  
Aojie Wang ◽  
Feng Zhou ◽  
Congcong Liu ◽  
Dongsheng Gao ◽  
Ruxi Qi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Immune Evasion ◽  
Cell Invasion ◽  
Structural Information ◽  
Host Cell Invasion ◽  
Hydrophobic Pocket ◽  
Viral Immune Evasion ◽  
Cryo Electron Microscopy ◽  
Reproductive Losses ◽  
Getah Virus

Getah virus (GETV) is a mosquito-borne pathogen that can cause a mild illness and reproductive losses in animals. Although antibodies to GETV have been found in humans, there are no reports of clinical symptom associated with GETV. However, antivirals or vaccine against GETV is still unavailable due to lack of knowledge of the structure of GETV virion. Here, we present the structure of mature GETV at a resolution of 2.8 Å with capsid protein, envelope glycoproteins E1 and E2. Glycosylation and S-acylation sites in E1 and E2 are identified. The surface-exposed glycans demonstrated their impact on the viral immune evasion and host cell invasion. The S-acylation sites strongly stabilize the virion. In addition, a cholesterol and phospholipid molecule are observed in transmembrane hydrophobic pocket, together with two more cholesterols surround the pocket. These structural information are helpful for structure-based antivirals and vaccine design.

Forge Wednesday Roundup - June 10, 2020

2021 ◽  
Author(s):  
H.L. Cu Si
Keyword(s):  
Electron Microscopy ◽  
Facial Recognition ◽  
Academic Research ◽  
Atomic Resolution ◽  
Rapa Nui ◽  
Social Distancing ◽  
Cryo Electron Microscopy

In today’s Roundup: COVID is still with us; pair of papers quantifies effects of social distancing; IBM bails on facial recognition, warns of harms; questioning “collapse” of Rapa Nui; attention turns to antibodies for COVID-19 therapies; WHO walks back confusing statement on asymptomatic COVID transmission; cryo-electron microscopy reveals proteins at atomic resolution; unstoppable newsbot runs amok, propagates bias; exploring the lure of misinformation; academic research goes on strike against racism; much more...

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