Cryo-EM and Single Particles

Physiology ◽  
2006 ◽  
Vol 21 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Liguo Wang ◽  
Fred J. Sigworth
Keyword(s):  
Electron Microscope ◽  
Membrane Protein ◽  
Single Particle ◽  
Protein Structures ◽  
Three Dimensional ◽  
Computer Processing ◽  
Two Dimensional ◽  
Single Particles ◽  
Macromolecular Assemblies ◽  
Particle Images

Cryoelectronmicroscopy is a method for the imaging of macromolecules in the electron microscope. It was originally developed to determine membrane protein structures from two-dimensional crystals, but more recently “single-particle” techniques have become powerful and popular. Three-dimensional reconstructions are obtained from sets of single-particle images by extensive computer processing; the methods are being applied to many macromolecular assemblies.

Computer Assisted Image Reconstruction of Oligomer Structure

1976 ◽  
Vol 34 ◽  
pp. 472-473
Author(s):  
A.M. Jones ◽  
A. Max Fiskin
Keyword(s):  
Three Dimensional ◽  
Depth Of Field ◽  
Computer Assisted ◽  
Projection Data ◽  
Two Dimensional ◽  
Single Particles ◽  
Dimensional Reconstruction ◽  
Computer Assisted Image ◽  
The Fourier Transform ◽  
Space Approach

If the tilt of a specimen can be varied either by the strategy of observing identical particles orientated randomly or by use of a eucentric goniometer stage, three dimensional reconstruction procedures are available (l). If the specimens, such as small protein aggregates, lack periodicity, direct space methods compete favorably in ease of implementation with reconstruction by the Fourier (transform) space approach (2). Regardless of method, reconstruction is possible because useful specimen thicknesses are always much less than the depth of field in an electron microscope. Thus electron images record the amount of stain in columns of the object normal to the recording plates. For single particles, practical considerations dictate that the specimen be tilted precisely about a single axis. In so doing a reconstructed image is achieved serially from two-dimensional sections which in turn are generated by a series of back-to-front lines of projection data.

Overabundant single-particle electron microscope views induce a three-dimensional reconstruction artifact

1998 ◽  
Vol 74 (4) ◽  
pp. 201-207 ◽  
Author(s):  
Nicolas Boisset ◽  
Pawel A. Penczek ◽  
Jean-Christophe Taveau ◽  
Valérie You ◽  
Felix de Haas ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Single Particle ◽  
Three Dimensional ◽  
Three Dimensional Reconstruction ◽  
Dimensional Reconstruction

Microcircuitry of the cat retina: Connections between arrays of neurons

1987 ◽  
Vol 45 ◽  
pp. 706-711
Author(s):  
Michael A. Freed ◽  
Peter Sterling
Keyword(s):  
Electron Microscope ◽  
Ganglion Cell ◽  
Ganglion Cells ◽  
Three Dimensional ◽  
Retinal Ganglion ◽  
Two Dimensional ◽  
Dimensional Electron ◽  
Rod Photoreceptors ◽  
Cat Retina ◽  
The Relationship

One of the most basic of structure-function relationships in the mammalian visual system is the relationship between the size of a ganglion cell's receptive field and the number of rod photoreceptors which are connected to it. There is also the flip side of the coin: how many ganglion cells does a single photoreceptor connect to? We have estimated the number of rods which converge upon an on-beta type of retinal ganglion cell; we have also estimated the number of on-beta ganglion cells which a single rod diverges to. Our method is to extract a three-dimensional circuit from a series of two-dimensional electron microscope sections. The results have implications for the preservation of the signal/noise ratio in the ganglion cell.There are two well-documented routes from the rods to the on-beta ganglion cell.

scholarly journals Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals

IUCrJ ◽  
2018 ◽  
Vol 5 (1) ◽  
pp. 103-117 ◽  
Author(s):  
Cecilia M. Casadei ◽  
Ching-Ju Tsai ◽  
Anton Barty ◽  
Mark S. Hunter ◽  
Nadia A. Zatsepin ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Large Scale ◽  
Structural Changes ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Single Layer ◽  
Protein Crystals ◽  
Single Shot ◽  
Two Dimensional ◽  
X Ray

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.

3D Reconstruction of Proteasomes Using Random Conical Tilting

1990 ◽  
Vol 48 (1) ◽  
pp. 272-273
Author(s):  
R. Hegerl ◽  
G. Pfeifer ◽  
B. Dahlmann ◽  
W. Baumeister
Keyword(s):  
3D Reconstruction ◽  
Tilt Angle ◽  
Three Dimensional ◽  
Ammonium Molybdate ◽  
Two Dimensional ◽  
Single Particles ◽  
Three Dimensional Model ◽  
Cylinder Axis ◽  
Electron Dose ◽  
Image Pairs

Proteasomes isolated from the archaebacterium Thermoplasma acidophilum and negatively stained with ammonium molybdate appear in two preferred orientations on electron micrographs. Describing the particle roughly as cylinder-shaped, the orientations "end-on" and "side-on" are defined by the cylinder axis being normal and parallel to the specimen support, respectively. Two-dimensional averages are available from both views. The complexity of the particle, however, prevents an intuitive deduction of a three-dimensional model from these two views. Due to the occurence of two preferred orientations, the object is, in principle, well suited for a 3D reconstruction based on random conical tilting. The side-on view was used for 3D reconstruction for two reasons: First, two-dimensional averages led us to suppose that the particles do not suffer from rotations around their cylinder axis, and second, parallelism of the long cylinder axis and the specimen support promises to give a stable object inclination.The proteasomes were isolated and prepared for electron microscopy as described previously. Many pairs of micrographs were taken in a Philips EM 420 at a magnification of 36000 and an electron dose of about 2000 e/nm2. The first exposure of each pair shows the specimen with a tilt angle of about 60 deg, the second one is untilted (Fig. 1). Several image pairs were selected for computer processing using a favourable distribution of particles and acceptable focus conditions as selection criteria. After digitizing corresponding areas (2048 by 2048 pixels, pixel size 15 μm = 0.42 nm at object level) in each pair using an Eikonix 1412 camera, small images of single particles were extracted from the untilted image and aligned with respect to translation and rotation. Knowing the tilt angle and tilt axis azimuth, position and rotation parameters of the particles could be transferred to the tilted image thus enabling the extraction of a set 227 of projections ready for a three-dimensional back projection (Fig. 2). All computation was done with the EM-system.

Realizing the potential of electron cryo-microscopy

2004 ◽  
Vol 37 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Richard Henderson
Keyword(s):  
Electron Microscopy ◽  
Structural Analysis ◽  
Electron Tomography ◽  
Biological Macromolecules ◽  
Two Dimensional ◽  
Future Prospects ◽  
Single Particles ◽  
Macromolecular Assemblies ◽  
Lower Symmetry ◽  
2D Crystals

1. Introduction 32. Background 53. 2D crystals 74. 1D crystals (helical arrays) 85. Icosahedral single particles 86. Single particles with lower symmetry 97. Cellular and subcellular electron tomography 108. Conclusion and future prospects 109. References 11Structural analysis by electron microscopy of biological macromolecules or macromolecular assemblies embedded in rapidly frozen, vitreous ice has made great advances during the last few years. Electron cryo-microscopy, or cryo-EM, can now be used to analyse the structures of molecules arranged in the form of two-dimensional crystals, helical arrays or as single particles with or without symmetry. Although it has been possible, using crystalline or helical specimens, to reach a resolution adequate to build atomic models (4 Å), there is every hope this will soon also be possible with single particles. Small and large single particles present different obstacles to progress.

scholarly journals Two-Dimensional and Three-Dimensional Single Particle Tracking of Upconverting Nanoparticles in Living Cells

2019 ◽  
Vol 20 (6) ◽  
pp. 1424 ◽  
Author(s):  
Kyujin Shin ◽  
Yo Song ◽  
Yeongchang Goh ◽  
Kang Lee
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Near Infrared ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Living Cells ◽  
Single Particle Tracking ◽  
Infrared Light ◽  
Objective Lens ◽  
Two Dimensional

Lanthanide-doped upconversion nanoparticles (UCNPs) are inorganic nanomaterials in which the lanthanide cations embedded in the host matrix can convert incident near-infrared light to visible or ultraviolet light. These particles are often used for long-term and real-time imaging because they are extremely stable even when subjected to continuous irradiation for a long time. It is now possible to image their movement at the single particle level with a scale of a few nanometers and track their trajectories as a function of time with a scale of a few microseconds. Such UCNP-based single-particle tracking (SPT) technology provides information about the intracellular structures and dynamics in living cells. Thus far, most imaging techniques have been built on fluorescence microscopic techniques (epifluorescence, total internal reflection, etc.). However, two-dimensional (2D) images obtained using these techniques are limited in only being able to visualize those on the focal planes of the objective lens. On the contrary, if three-dimensional (3D) structures and dynamics are known, deeper insights into the biology of the thick cells and tissues can be obtained. In this review, we introduce the status of the fluorescence imaging techniques, discuss the mathematical description of SPT, and outline the past few studies using UCNPs as imaging probes or biologically functionalized carriers.

scholarly journals AN ELECTRON MICROSCOPE EXAMINATION OF URINARY MUCOPROTEIN AND ITS INTERACTION WITH INFLUENZA VIRUS

1964 ◽  
Vol 21 (2) ◽  
pp. 265-274 ◽  
Author(s):  
M. E. Bayer
Keyword(s):  
Heat Treatment ◽  
Influenza Virus ◽  
Electron Microscope ◽  
Human Urine ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Microscope Examination ◽  
Virus Particles ◽  
Single Branch ◽  
Complicated Configuration

A hemagglutination-inhibitory mucoprotein from human urine has been studied with the electron microscope. It consists of filaments, with diameters of 40 to > 240 A, composed of smaller fibrils. In the two-dimensional projection of the electron micrographs, the single fibrils often show a zig-zag course with a periodicity of 100 to 140 A; the single branch of a zig-zag measures about 60 A in length and either 20 or 40 A in width. Still thinner fibrillar elements are observable with diameters of 10 A or less. In three-dimensional aspect, the zig-zag structure might be a helix. The fibril-bundle (or filament) reveals a complicated configuration. Heat treatment at 70°C shows some indication of denaturation (e.g. filaments are shorter), whereas at 80°C almost complete degradation of the protein into individual zig-zag elements or smaller pieces is attained. The interaction between influenza virus particles and inhibitory mucoprotein consists of the attachment of a fiber molecule to the virus projections at several sites and frequently on more than one virus particle.

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