electron crystallography
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Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.
Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) play a crucial role in structure-guided drug discovery and design, and also provide atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. The quality with which small-molecule ligands have been modelled in Protein Data Bank (PDB) entries has been, and continues to be, a matter of concern for many investigators. Correctly interpreting whether electron density found in a binding site is compatible with the soaked or co-crystallized ligand or represents water or buffer molecules is often far from trivial. The Worldwide PDB validation report (VR) provides a mechanism to highlight any major issues concerning the quality of the data and the model at the time of deposition and annotation, so the depositors can fix issues, resulting in improved data quality. The ligand-validation methods used in the generation of the current VRs are described in detail, including an examination of the metrics to assess both geometry and electron-density fit. It is found that the LLDF score currently used to identify ligand electron-density fit outliers can give misleading results and that better ligand-validation metrics are required.
- MeSH
- databáze proteinů * MeSH
- konformace proteinů * MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- ligandy MeSH
- makromolekulární látky chemie MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- proteiny analýza chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
... The basic principles of the electron microscope 1 -- 1.1 The use of electrons for microscopy 2 -- 1.2 ... ... Electron lenses 4 -- 1.3 Defects of electron lenses 9 -- 1.3.1 Spherical aberration 9 -- 1.3.2 Chromatic ... ... The design of the electron microscope 38 -- 2.1 General construction of the column 38 -- 2.2 The electron ... ... microscope 110 -- 3.10.1 Electron diffraction 110 -- 3.10.2 High dispersion electron diffraction 112 ... ... image 267 -- 7.8 Electronic display and recording systems 268 -- 7.9 Viewing the electron image 270 ...
Practical methods in electron microscopy ; v. 2
Laboratory edition xii, 345 stran : ilustrace ; 23 cm
- MeSH
- elektronová mikroskopie MeSH
- fyzika MeSH
- mikroskopie MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Fyzika
- NLK Obory
- fyzika, biofyzika
A method has been developed that automatically fits double-helical regions into the electron density of nucleic acid structures. Rigid fragments consisting of two Watson-Crick base pairs and three pairs of phosphate groups in the A-type or B-type conformation are positioned into the electron density by phased rotation and translation functions. The position and orientation of the localized double-helical fragments are determined by phased refinement. The method has been tested by building double-helical regions of nine RNA structures of variable crystallographic resolution and polynucleotide length and is available for free use.
Crystallography has long been the unrivaled method that can provide the atomistic structural models of macromolecules, using either X-rays or electrons as probes. The methodology has gone through several revolutionary periods, driven by the development of new sources, detectors, and other instrumentation. Novel sources of both X-ray and electrons are constantly emerging. The increase in brightness of these sources, complemented by the advanced detection techniques, has relaxed the traditionally strict need for large, high quality, crystals. Recent reports suggest high-quality diffraction datasets from crystals as small as a few hundreds of nanometers can be routinely obtained. This has resulted in the genesis of a new field of macromolecular nanocrystal crystallography. Here we will make a brief comparative review of this growing field focusing on the use of X-rays and electrons sources.
The impact of structural biology on drug discovery is well documented, and the workhorse technique for the past 30 years or so has been X-ray crystallography. With the advent of several technological improvements, including direct electron detectors, automation, better microscope vacuums and lenses, phase plates and improvements in computing power enabled by GPUs, it is now possible to record and analyse images of protein structures containing high-resolution information. This review, from a pharmaceutical perspective, highlights some of the most relevant and interesting protein structures for the pharmaceutical industry and shows examples of how ligand-binding sites, membrane proteins, both big and small, pseudo symmetry and complexes are being addressed by this technique.
- MeSH
- buněčné inkluze virové MeSH
- buněčné jádro mikrobiologie MeSH
- cytopatogenní efekt virový MeSH
- cytoplazma mikrobiologie MeSH
- elektronová mikroskopie MeSH
- HeLa buňky MeSH
- Herpesviridae růst a vývoj MeSH
- krystalografie MeSH
- prasečí herpesvirus 1 patogenita růst a vývoj ultrastruktura MeSH
- replikace viru MeSH
- virulence MeSH
UNLABELLED: In order to initiate an infection, viruses need to deliver their genomes into cells. This involves uncoating the genome and transporting it to the cytoplasm. The process of genome delivery is not well understood for nonenveloped viruses. We address this gap in our current knowledge by studying the uncoating of the nonenveloped human cardiovirus Saffold virus 3 (SAFV-3) of the family Picornaviridae SAFVs cause diseases ranging from gastrointestinal disorders to meningitis. We present a structure of a native SAFV-3 virion determined to 2.5 Å by X-ray crystallography and an 11-Å-resolution cryo-electron microscopy reconstruction of an "altered" particle that is primed for genome release. The altered particles are expanded relative to the native virus and contain pores in the capsid that might serve as channels for the release of VP4 subunits, N termini of VP1, and the RNA genome. Unlike in the related enteroviruses, pores in SAFV-3 are located roughly between the icosahedral 3- and 5-fold axes at an interface formed by two VP1 and one VP3 subunit. Furthermore, in native conditions many cardioviruses contain a disulfide bond formed by cysteines that are separated by just one residue. The disulfide bond is located in a surface loop of VP3. We determined the structure of the SAFV-3 virion in which the disulfide bonds are reduced. Disruption of the bond had minimal effect on the structure of the loop, but it increased the stability and decreased the infectivity of the virus. Therefore, compounds specifically disrupting or binding to the disulfide bond might limit SAFV infection. IMPORTANCE: A capsid assembled from viral proteins protects the virus genome during transmission from one cell to another. However, when a virus enters a cell the virus genome has to be released from the capsid in order to initiate infection. This process is not well understood for nonenveloped viruses. We address this gap in our current knowledge by studying the genome release of Human Saffold virus 3 Saffold viruses cause diseases ranging from gastrointestinal disorders to meningitis. We show that before the genome is released, the Saffold virus 3 particle expands, and holes form in the previously compact capsid. These holes serve as channels for the release of the genome and small capsid proteins VP4 that in related enteroviruses facilitate subsequent transport of the virus genome into the cell cytoplasm.
Bacteriophage ϕ6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ϕ6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C2221. Matthews's coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ϕ6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.
- MeSH
- bakteriofág phi 6 chemie MeSH
- elektronová kryomikroskopie MeSH
- konformace proteinů MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- virové plášťové proteiny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
