Two-Dimensional Simplification of Complex Three-Dimensional Wire Mesh Screens

This tutorial will discuss the methodology of low dose electron diffraction and imaging of crystalline biological objects, the problems of data interpretation for two-dimensional projected density maps of glucose embedded protein crystals, the factors to be considered in combining tilt data from three-dimensional crystals, and finally, the prospects of achieving a high resolution three-dimensional density map of a biological crystal. This methodology will be illustrated using two proteins under investigation in our laboratory, the T4 DNA helix destabilizing protein gp32*I and the crotoxin complex crystal.

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Instrumentation For Quantitative Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100078584 ◽

1977 ◽

Vol 35 ◽

pp. 206-209

Author(s):

B. Ralph ◽

A.R. Jones

Keyword(s):

Volume Fraction ◽

Three Dimensional ◽

Two Dimensional ◽

Automatic Data ◽

Phase Volume Fraction ◽

Statistical Confidence ◽

Resultant Data ◽

Automatic Data Collection ◽

Third Dimension ◽

Evolution Of Microstructure

In all fields of microscopy there is an increasing interest in the quantification of microstructure. This interest may stem from a desire to establish quality control parameters or may have a more fundamental requirement involving the derivation of parameters which partially or completely define the three dimensional nature of the microstructure. This latter categorey of study may arise from an interest in the evolution of microstructure or from a desire to generate detailed property/microstructure relationships. In the more fundamental studies some convolution of two-dimensional data into the third dimension (stereological analysis) will be necessary.In some cases the two-dimensional data may be acquired relatively easily without recourse to automatic data collection and further, it may prove possible to perform the data reduction and analysis relatively easily. In such cases the only recourse to machines may well be in establishing the statistical confidence of the resultant data. Such relatively straightforward studies tend to result from acquiring data on the whole assemblage of features making up the microstructure. In this field data mode, when parameters such as phase volume fraction, mean size etc. are sought, the main case for resorting to automation is in order to perform repetitive analyses since each analysis is relatively easily performed.

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A linearity test for thick specimens imaged by HVEM over a 180° angular range

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100087070 ◽

1991 ◽

Vol 49 ◽

pp. 552-553

Author(s):

Yu Liu

Keyword(s):

Phase Contrast ◽

Three Dimensional ◽

Angular Range ◽

Two Dimensional ◽

Back Projection ◽

Line Integral ◽

Exponential Law ◽

Transmission Electron ◽

Absorption Law ◽

Linearity Test

The image obtained in a transmission electron microscope is the two-dimensional projection of a three-dimensional (3D) object. The 3D reconstruction of the object can be calculated from a series of projections by back-projection, but this algorithm assumes that the image is linearly related to a line integral of the object function. However, there are two kinds of contrast in electron microscopy, scattering and phase contrast, of which only the latter is linear with the optical density (OD) in the micrograph. Therefore the OD can be used as a measure of the projection only for thin specimens where phase contrast dominates the image. For thick specimens, where scattering contrast predominates, an exponential absorption law holds, and a logarithm of OD must be used. However, for large thicknesses, the simple exponential law might break down due to multiple and inelastic scattering.

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An Image Reconstruction System Applied to High Voltage Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115080 ◽

1975 ◽

Vol 33 ◽

pp. 292-293

Author(s):

D. E. Johnson

Keyword(s):

High Voltage ◽

Prior Information ◽

Block Diagram ◽

Three Dimensional ◽

Real Space ◽

Two Dimensional ◽

Efficient Manner ◽

Fourier Methods ◽

Tilt Series ◽

Methods Of Reconstruction

Increased specimen penetration; the principle advantage of high voltage microscopy, is accompanied by an increased need to utilize information on three dimensional specimen structure available in the form of two dimensional projections (i.e. micrographs). We are engaged in a program to develop methods which allow the maximum use of information contained in a through tilt series of micrographs to determine three dimensional speciman structure.In general, we are dealing with structures lacking in symmetry and with projections available from only a limited span of angles (±60°). For these reasons, we must make maximum use of any prior information available about the specimen. To do this in the most efficient manner, we have concentrated on iterative, real space methods rather than Fourier methods of reconstruction. The particular iterative algorithm we have developed is given in detail in ref. 3. A block diagram of the complete reconstruction system is shown in fig. 1.

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Computer Assisted Image Reconstruction of Oligomer Structure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092323 ◽

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.

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Polymorphic phase transition of a membrane protein - analysis of continuous diffuse electron diffraction intensity

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142451 ◽

1986 ◽

Vol 44 ◽

pp. 166-167

Author(s):

Douglas L. Dorset ◽

Andrew K. Massalski

Keyword(s):

Molecular Sieve ◽

Three Dimensional ◽

Pore Geometry ◽

Two Dimensional ◽

Diffraction Intensity ◽

Polymorphic Phase Transition ◽

E Coli ◽

Crystallographic Study ◽

Hexagonal Form ◽

Polymorphic Forms

Matrix porin, the ompF gene product of E. coli, has been the object of a electron crystallographic study of its pore geometry in an attempt to understand its function as a membrane molecular sieve. Three polymorphic forms have been found for two-dimensional crystals reconstituted in phospholipid, two hexagonal forms with different lipid content and an orthorhombic form coexisting with and similar to the hexagonal form found after lipid loss. In projection these have been shown to retain the same three-fold pore triplet geometry and analyses of three-dimensional data reveal that the small hexagonal and orthorhombic polymorphs have similar structure as well as unit cell spacings.

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Two-Dimensional Crystallization of Tetanus Toxin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119466 ◽

1985 ◽

Vol 43 ◽

pp. 528-529

Author(s):

Jeffry A. Reidler ◽

John P. Robinson

Keyword(s):

Electron Microscopy ◽

Tetanus Toxin ◽

Structural Information ◽

Three Dimensional ◽

Two Dimensional ◽

Membrane Interface ◽

3D Reconstructions ◽

Cellular Receptors ◽

Computer Reconstruction ◽

2D Crystals

We have prepared two-dimensional (2D) crystals of tetanus toxin using procedures developed by Uzgiris and Kornberg for the directed production of 2D crystals of monoclonal antibodies at an antigen-phospholipid monolayer interface. The tetanus toxin crystals were formed using a small mole fraction of the natural receptor, GT1, incorporated into phosphatidyl choline monolayers. The crystals formed at low concentration overnight. Two dimensional crystals of this type are particularly useful for structure determination using electron microscopy and computer image refinement. Three dimensional (3D) structural information can be derived from these crystals by computer reconstruction of photographs of toxin crystals taken at different tilt angles. Such 3D reconstructions may help elucidate the mechanism of entry of the enzymatic subunit of toxins into cells, particularly since these crystals form directly on a membrane interface at similar concentrations of ganglioside GT1 to the natural cellular receptors.

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Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180161 ◽

1990 ◽

Vol 48 (1) ◽

pp. 282-283

Author(s):

José L. Carrascosa ◽

José M. Valpuesta ◽

Hisao Fujisawa

Keyword(s):

Dna Sequences ◽

Expression Vector ◽

Three Dimensional ◽

Deae Cellulose ◽

Dna Packaging ◽

Dimensional Structure ◽

Two Dimensional ◽

Freezing And Thawing ◽

Three Dimensional Structure ◽

Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

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Three-Dimensional Reconstruction of Two Forms of the Chaperonin GRO EL

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180045 ◽

1990 ◽

Vol 48 (1) ◽

pp. 258-259

Author(s):

J.L. Carrascosa ◽

G. Abella ◽

S. Marco ◽

M. Muyal ◽

J.M. Carazo

Keyword(s):

Three Dimensional ◽

The Other ◽

Two Dimensional ◽

Series Method ◽

Three Dimensional Reconstruction ◽

Structural Components ◽

E Coli ◽

Dimensional Reconstruction ◽

Morphogenetic Factors ◽

Tilt Series

Chaperonins are a class of proteins characterized by their role as morphogenetic factors. They trantsiently interact with the structural components of certain biological aggregates (viruses, enzymes etc), promoting their correct folding, assembly and, eventually transport. The groEL factor from E. coli is a conspicuous member of the chaperonins, as it promotes the assembly and morphogenesis of bacterial oligomers and/viral structures.We have studied groEL-like factors from two different bacteria:E. coli and B.subtilis. These factors share common morphological features , showing two different views: one is 6-fold, while the other shows 7 morphological units. There is also a correlation between the presence of a dominant 6-fold view and the fact of both bacteria been grown at low temperature (32°C), while the 7-fold is the main view at higher temperatures (42°C). As the two-dimensional projections of groEL were difficult to interprete, we studied their three-dimensional reconstruction by the random conical tilt series method from negatively stained particles.

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