Editorial overview: Cryo electron microscopy: The impact of the cryo-EM revolution in biology

2018 ◽  
Vol 52 ◽  
pp. iii-v
Keyword(s):  
Electron Microscopy ◽  
Cryo Electron Microscopy ◽  
The Impact

scholarly journals Using cryo-EM to uncover mechanisms of bacterial transcriptional regulation

2021 ◽  
Author(s):  
David M. Wood ◽  
Renwick C.J. Dobson ◽  
Christopher R. Horne
Keyword(s):  
Electron Microscopy ◽  
Transcriptional Regulation ◽  
Cellular Response ◽  
Target Genes ◽  
Control Point ◽  
Structural Investigations ◽  
Bacterial Gene ◽  
Cryo Electron Microscopy ◽  
Transcription Complexes ◽  
The Impact

Transcription is the principal control point for bacterial gene expression, and it enables a global cellular response to an intracellular or environmental trigger. Transcriptional regulation is orchestrated by transcription factors, which activate or repress transcription of target genes by modulating the activity of RNA polymerase. Dissecting the nature and precise choreography of these interactions is essential for developing a molecular understanding of transcriptional regulation. While the contribution of X-ray crystallography has been invaluable, the ‘resolution revolution’ of cryo-electron microscopy has transformed our structural investigations, enabling large, dynamic and often transient transcription complexes to be resolved that in many cases had resisted crystallisation. In this review, we highlight the impact cryo-electron microscopy has had in gaining a deeper understanding of transcriptional regulation in bacteria. We also provide readers working within the field with an overview of the recent innovations available for cryo-electron microscopy sample preparation and image reconstruction of transcription complexes.

Using Cryo-EM to Investigate Bacterial Secretion Systems

2018 ◽  
Vol 72 (1) ◽  
pp. 231-254 ◽  
Author(s):  
Chiara Rapisarda ◽  
Matteo Tassinari ◽  
Francesca Gubellini ◽  
Rémi Fronzes
Keyword(s):  
Electron Microscopy ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Secretion Systems ◽  
Extracellular Milieu ◽  
Cryo Electron Microscopy ◽  
Bacterial Fitness ◽  
High Resolution Images ◽  
The Impact ◽  
Bacterial Secretion

Bacterial secretion systems are responsible for releasing macromolecules to the extracellular milieu or directly into other cells. These membrane complexes are associated with pathogenicity and bacterial fitness. Understanding of these large assemblies has exponentially increased in the last few years thanks to electron microscopy. In fact, a revolution in this field has led to breakthroughs in characterizing the structures of secretion systems and other macromolecular machineries so as to obtain high-resolution images of complexes that could not be crystallized. In this review, we give a brief overview of structural advancements in the understanding of secretion systems, focusing in particular on cryo–electron microscopy, whether tomography or single-particle analysis. We describe how such techniques have contributed to knowledge of the mechanism of macromolecule secretion in bacteria and the impact they will have in the future.

scholarly journals High-resolution cryo-EM structure of urease from the pathogen Yersinia enterocolitica

2020 ◽  
Author(s):  
Ricardo D. Righetto ◽  
Leonie Anton ◽  
Ricardo Adaixo ◽  
Roman P. Jakob ◽  
Jasenko Zivanov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Carbon Dioxide ◽  
High Resolution ◽  
Yersinia Enterocolitica ◽  
Active Site ◽  
High Data ◽  
Cryo Electron Microscopy ◽  
Acidic Environments ◽  
The Impact ◽  
Better Than

AbstractUrease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at better than 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. Our data are of sufficient quality to support drug development efforts.

scholarly journals High-resolution cryo-EM structure of urease from the pathogen Yersinia enterocolitica

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ricardo D. Righetto ◽  
Leonie Anton ◽  
Ricardo Adaixo ◽  
Roman P. Jakob ◽  
Jasenko Zivanov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Carbon Dioxide ◽  
High Resolution ◽  
Drug Development ◽  
Yersinia Enterocolitica ◽  
Active Site ◽  
High Data ◽  
Cryo Electron Microscopy ◽  
Acidic Environments ◽  
The Impact

AbstractUrease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. The obtained structure is of sufficient quality to support drug development efforts.

scholarly journals The mechanism of translation

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 198 ◽  
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Recent Literature ◽  
Binding Modes ◽  
Recording Medium ◽  
X Ray ◽  
The Past ◽  
Cryo Electron Microscopy ◽  
Work Cycle ◽  
The Impact

Translation of the genetic code on the ribosome into protein is a process of extraordinary complexity, and understanding its mechanism has remained one of the major challenges even though x-ray structures have been available since 2000. In the past two decades, single-particle cryo-electron microscopy has contributed a major share of information on structure, binding modes, and conformational changes of the ribosome during its work cycle, but the contributions of this technique in the translation field have recently skyrocketed after the introduction of a new recording medium capable of detecting individual electrons. As many examples in the recent literature over the past three years show, the impact of this development on the advancement of knowledge in this field has been transformative and promises to be lasting.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

Study of eukaryotic flagellar components by cryo-electron microscopy

1986 ◽  
Vol 44 ◽  
pp. 98-101
Author(s):  
John M. Murray ◽  
Rob Ward
Keyword(s):  
Electron Microscopy ◽  
Primary Motion ◽  
Cryo Electron Microscopy ◽  
Cross Links ◽  
Cilia And Flagella

The eukaryotic flagellum is constructed from 11 parallel tubular elements arranged as 9 peripheral fibers (doublet microtubules) and 2 central fibers (singlet microtubules). The primary motion generating component has been found to be arranged as axially periodic “arms” bridging the adjacent doublets. The dynein, comprising the arms, has been isolated and characterized from several different cilia and flagella. Various radial and azimuthal cross-links stabilize the axially aligned microtubules, and probably play some role in controlling the form of the flagella beat cycle.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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