Electron Diffraction with the Transmission Electron Microscope as a Phase‐Determining Diffractometer—From Spatial Frequency Filtering to the Three‐Dimensional Structure Analysis of Ribosomes

Hoppe, W.

doi:10.1002/anie.198304561

Cited by 20 publications

(5 citation statements)

References 81 publications

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“…In contrast, the EM produces actual images, which is tantamount to saying it measures both amplitudes and phases in Fourier space, giving rise to an adage coined by Walter Hoppe’s (see Hoppe, 1983), of the EM as a ‘phase-measuring diffractometer.’…”

Section: Single Molecules Versus Crystalsmentioning

confidence: 99%

“…In X-ray crystallography, information on amplitudes is concentrated in small spots or layer lines in the Fourier transform, and phases are not supplied directly by the diffractometer but have to be garnered by computation with the aid of additional experiments, employing multiple heavy atom replacements or multiple wavelengths. In contrast, the EM produces actual images, which is tantamount to saying it measures both amplitudes and phases in Fourier space, giving rise to an adage coined by Walter Hoppe's (see Hoppe, 1983), of the EM as a ‘phase-measuring diffractometer.’…”

Section: Single Molecules Versus Crystalsmentioning

confidence: 99%

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Single-particle reconstruction of biological macromolecules in electron microscopy – 30 years

Frank

2009

Quart. Rev. Biophys.

130

108

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This essay gives the author's personal account on the development of concepts underlying singleparticle reconstruction, a technique in electron microscopy of macromolecular assemblies with a remarkable record of achievements as of late. The ribosome proved to be an ideal testing ground for the development of specimen preparation methods, cryo-EM techniques, and algorithms, with discoveries along the way as a rich reward. Increasingly, cryo-EM and single-particle reconstruction, in combination with classification techniques, is revealing dynamic information on functional molecular machines uninhibited by molecular contacts.

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Section: Single Molecules Versus Crystalsmentioning

confidence: 99%

Section: Single Molecules Versus Crystalsmentioning

confidence: 99%

Single-particle reconstruction of biological macromolecules in electron microscopy – 30 years

Frank

2009

Quart. Rev. Biophys.

130

108

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“…The review does not include any consideration of near-field microscopy using scanning probes of any kind, nor methods which only look at surfaces. Previous publications which have considered the relative merits of neutron, X-ray or electron microscopy include Breedlove & Trammel (1970), Hoppe (1983) and Sayre et al (1977). Useful text books include Cowley (1975), Reimer (1989) and Spence (1988).…”

Section: Introductionmentioning

confidence: 99%

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules

Henderson

1995

Quart. Rev. Biophys.

1,107

890

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Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associated energy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of approximately 10(5) molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10,000 particles are required. Below 10(5) molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

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“…2.3 and 2.4 of Natesh [3]) does not exist in EM. The electron microscope, in Hoppe's words, is a "phase-measuring diffractometer" [40]. Hence, extreme care has to be taken in image processing.…”

Section: Image Processing and Three-dimensional Reconstructionmentioning

confidence: 99%

Single-Particle cryo-EM as a Pipeline for Obtaining Atomic Resolution Structures of Druggable Targets in Preclinical Structure-Based Drug Design

Natesh

2019

Challenges and Advances in Computational Chemistry and Physics

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Single-particle cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image processing have gained importance in the last few years to obtain atomic structures of drug targets. Obtaining atomic-resolution 3D structure better than *2.5 Å is a standard approach in pharma companies to design and optimize therapeutic compounds against drug targets like proteins. Protein crystallography is the main technique in solving the structures of drug targets at atomic resolution. However, this technique requires protein crystals which in turn is a major bottleneck. It was not possible to obtain the structure of proteins better than 2.5 Å resolution by any other methods apart from protein crystallography until 2015. Recent advances in single-particle cryo-EM and 3D image processing have led to a resolution revolution in the field of structural biology that has led to high-resolution protein structures, thus breaking the cryo-EM resolution barriers to facilitate drug discovery. There are 24 structures solved by single-particle cryo-EM with resolution 2.5 Å or better in the EMDataBank (EMDB) till date. Among these, five cryo-EM 3D reconstructions of proteins in the EMDB have their associated coordinates deposited in Protein Data Bank (PDB), with bound inhibitor/ ligand. Thus, for the first time, single-particle cryo-EM was included in the structure-based drug design (SBDD) pipeline for solving protein structures independently or where crystallography has failed to crystallize the protein. Further, this technique can be complementary and supplementary to protein crystallography field in solving 3D structures. Thus, single-particle cryo-EM can become a standard approach in pharmaceutical industry in the design, validation, and optimization of therapeutic compounds targeting therapeutically important protein molecules during preclinical drug discovery research. The present chapter will describe briefly the history and the principles of single-particle cryo-EM and 3D image processing to obtain atomic-resolution structure of proteins and their complex with their drug targets/ligands.

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Electron Diffraction with the Transmission Electron Microscope as a Phase‐Determining Diffractometer—From Spatial Frequency Filtering to the Three‐Dimensional Structure Analysis of Ribosomes

Cited by 20 publications

References 81 publications

Single-particle reconstruction of biological macromolecules in electron microscopy – 30 years

Single-particle reconstruction of biological macromolecules in electron microscopy – 30 years

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules

Single-Particle cryo-EM as a Pipeline for Obtaining Atomic Resolution Structures of Druggable Targets in Preclinical Structure-Based Drug Design

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