scholarly journals Elucidation of fibril structure responsible for swimming in Spiroplasma using electron microscopy

2021 ◽  
Author(s):  
Yuya Sasajima ◽  
Takayuki Kato ◽  
Tomoko Miyata ◽  
Keiichi Namba ◽  
Makoto Miyata
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Ring Structure ◽  
Particle Analysis ◽  
Flagellar Motility ◽  
Research Attention ◽  
Filament Axis ◽  
Fibril Structure ◽  
Left And Right

AbstractSpiroplasma, known pathogens of arthropods and plants, are helical-shaped bacteria lacking the peptidoglycan layer. They swim by alternating between left- and right-handed cell helicity, which is driven by an internal structure called the ribbon. This system is unrelated to flagellar motility that is widespread in bacteria. The ribbon comprises the bacterial actin homolog MreB and fibril, the protein specific to Spiroplasma. Here, we isolated the ribbon and its core, the fibril filament, and using electron microscopy, found that the helicity of the ribbon and the cell is linked to the helicity of the fibril. Single particle analysis using the negative-staining method revealed that the three-dimensional structures of the fibril filament comprise a repeated ring structure twisting along the filament axis. Based on these observations, we propose a scheme for the helicity-switching mechanism in which the twists caused by the conformational changes in the fibril filament are accumulated, transmitted to the ribbon, and then propel the cells by rotating the cell body like a screw.Significance StatementSpiroplasma are widespread globally as pathogens of animals and plants. They are also recognized as male-killing bacteria of insects. Their special swimming mechanism is caused by helicity switching, which could be the simplest swimming mechanism. This mechanism has attracted research attention for many years because of the possible application in the field of nano actuators; however, the details of this mechanism remain to be clarified. Here, we reveal the outline of the swimming mechanism by analyzing the structure of the core of the Spiroplasma ribbon.

scholarly journals Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy

2019 ◽  
Vol 75 (1) ◽  
pp. 19-32 ◽  
Author(s):  
C. O. S. Sorzano ◽  
A. Jiménez ◽  
J. Mota ◽  
J. L. Vilas ◽  
D. Maluenda ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Image Processing ◽  
High Resolution ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
Conformational Variability ◽  
Established Technique

Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

scholarly journals A Survey of the Use of Iterative Reconstruction Algorithms in Electron Microscopy

2017 ◽  
Vol 2017 ◽  
pp. 1-17 ◽  
Author(s):  
C. O. S. Sorzano ◽  
J. Vargas ◽  
J. Otón ◽  
J. M. de la Rosa-Trevín ◽  
J. L. Vilas ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Tomographic Reconstruction ◽  
Three Dimensional ◽  
Iterative Algorithms ◽  
Projection Methods ◽  
Particle Analysis ◽  
Reconstruction Algorithms ◽  
Large Families ◽  
Key Steps

One of the key steps in Electron Microscopy is the tomographic reconstruction of a three-dimensional (3D) map of the specimen being studied from a set of two-dimensional (2D) projections acquired at the microscope. This tomographic reconstruction may be performed with different reconstruction algorithms that can be grouped into several large families: direct Fourier inversion methods, back-projection methods, Radon methods, or iterative algorithms. In this review, we focus on the latter family of algorithms, explaining the mathematical rationale behind the different algorithms in this family as they have been introduced in the field of Electron Microscopy. We cover their use in Single Particle Analysis (SPA) as well as in Electron Tomography (ET).

scholarly journals The Resolution in X-ray Crystallography and Single-Particle Cryogenic Electron Microscopy

Crystals ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 580
Author(s):  
Victor R.A. Dubach ◽  
Albert Guskov
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Fourier Transform

X-ray crystallography and single-particle analysis cryogenic electron microscopy are essential techniques for uncovering the three-dimensional structures of biological macromolecules. Both techniques rely on the Fourier transform to calculate experimental maps. However, one of the crucial parameters, resolution, is rather broadly defined. Here, the methods to determine the resolution in X-ray crystallography and single-particle analysis are summarized. In X-ray crystallography, it is becoming increasingly more common to include reflections discarded previously by traditionally used standards, allowing for the inclusion of incomplete and anisotropic reflections into the refinement process. In general, the resolution is the smallest lattice spacing given by Bragg’s law for a particular set of X-ray diffraction intensities; however, typically the resolution is truncated by the user during the data processing based on certain parameters and later it is used during refinement. However, at which resolution to perform such a truncation is not always clear and this makes it very confusing for the novices entering the structural biology field. Furthermore, it is argued that the effective resolution should be also reported as it is a more descriptive measure accounting for anisotropy and incompleteness of the data. In single particle cryo-EM, the situation is not much better, as multiple ways exist to determine the resolution, such as Fourier shell correlation, spectral signal-to-noise ratio and the Fourier neighbor correlation. The most widely accepted is the Fourier shell correlation using a threshold of 0.143 to define the resolution (so-called “gold-standard”), although it is still debated whether this is the correct threshold. Besides, the resolution obtained from the Fourier shell correlation is an estimate of varying resolution across the density map. In reality, the interpretability of the map is more important than the numerical value of the resolution.

scholarly journals A national facility for biological cryo-electron microscopy

2015 ◽  
Vol 71 (1) ◽  
pp. 127-135 ◽  
Author(s):  
Helen R. Saibil ◽  
Kay Grünewald ◽  
David I. Stuart
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Dimensional Electron ◽  
Cryo Electron Microscopy ◽  
Cellular Context ◽  
Molecular Machinery ◽  
User Friendly ◽  
Integrated Structural Biology ◽  
Expert Support

Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.

Structural Organization of Detergent-Extracted Cells

1980 ◽  
Vol 38 ◽  
pp. 814-817
Author(s):  
Manfred Schliwa
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Conformational Changes ◽  
Structural Organization ◽  
Three Dimensional ◽  
High Voltage Electron Microscopy ◽  
Dimensional Lattice ◽  
Voltage Electron ◽  
Cell Architecture ◽  
Critical Point Drying

Adequate visualization of the three-dimensional organization has always been a major problem in studies of cell architecture. Efforts of numerous investigators weredevoted to the question of how best information can be collected from specimens prepared with different procedures. In recent years, the potential of high voltage electron microscopy has been combined with a technique for sample preparation that circumvents embedding, namely critical point-drying from CO2, to study the three-dimensional fine structure of cells in culture. This approach has revealed new insights into the structural organization of the cytoplasm (1-4). A system of slender strands or microtrabeculae has been described to form an elaborate three-dimensional lattice in which other organelles are embedded. This system has been shown in some cells to undergo rapid conformational changes (3,5) and in general is believed to be an important component of the cytoskeleton, being responsible for the gelatious properties of the cytoplasm.

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