2,108,382 research outputs found
High-throughput high-resolution cryo-electron crystallography
Get PDFHigh-resolution structures of membrane and soluble proteins can be obtained by cryo-electron crystallography, given highly-ordered cryo-preparations of perfectly flat 2D crystals are available. Studies of membrane proteins, which are embedded into a lipid membrane, mimicking the native cell membrane, are of particular biological interest. However there are multiple reasons why electron crystallography is far from being a mainstream protein structure determination technique. In this thesis we address three major difficulties of electron crystallography: (i) resolution loss due to not perfectly flat crystals, (ii) reliable high-throughput automatic image processing and (iii) correction of electron beam-induced motion of the sample.
The conventional electron crystallography image processing procedure assumes perfectly flat 2D crystals, which are almost impossible to obtain. Our new processing approach, described in Chapter 2, weakens this assumption tremendously. Traditional processing assigns the same tilt geometry to all proteins of one 2D crystal. Thus local tilt geometry variations, due to not perfectly flat crystals, are neglected. We developed an algorithm that optimizes the tilt geometry of each protein separately, while exploiting the correlation between neighboring proteins. The new method proves the feasibility of this approach, improves the achieved resolution and opens the doors to new studies, i.e. structural studies of membrane proteins embedded into lipid vesicles.
Recently a new generation of digital detectors tremendously changed the cryo-electron microscopy field. Beside a significantly increased signal-to-noise ratio these detectors record dose-fractionated movies of the sample under the electron beam. Previous cameras only recorded one image instead. This new exposure mode enables the computational correction for beam-induced sample movements. In Chapter 3 we describe a new “real-time” automation pipeline for electron crystallography using direct electron detectors. The novel pipeline automatically corrects for homogeneous sample drift on frame level and processes the acquired images automatically. Both, the time-to-solution and the quality of the obtained 3D reconstructions are significantly improved.
Heterogeneous beam-induced sample movements are the most severe resolution- limiting factor in modern cryo-electron crystallography. In Chapter 4 we present an algorithm, termed movie-mode unbending, which corrects for inhomogeneous beam- induced sample drift. In contrast to the previous homogeneous drift-correction, the novel algorithm can correct for locally varying beam-induced sample motion. This novel approach significantly increases the resolution of electron cryo-crystallographic studies recorded on the latest detectors.
Chapter 5 covers multiple successful applications of the methods introduced above. For instance the real-time drift-correction module of the developed automation pipeline was used for multiple near-atomic resolution single particle and helical image processing projects. The high-throughput crystal image processing was applied to different kinds of 2D crystals and enabled the qualitative assessment of different sample preparation methods. Additionally we describe the implementation of a high-throughput single particle automation pipeline, which will enable the generation of near-atomic resolution single particle cryo-electron microscopy density maps on a daily basis
Mineralogical Crystallography
No full textCrystallography remains, for mineralogy, one of the main sources of information on natural crystalline substances [...
Nanoflow electrospinning serial femtosecond crystallography
No full textAn electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 µl min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min(-1) and diffracted to beyond 4 Å resolution, producing 14,000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.</p
Radiation damage to nucleoprotein complexes in macromolecular crystallography
Get PDFSignificant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein-DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07-44.63MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1C and sugar-phosphate CO bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses
<i>Journal of Applied Crystallography</i>: the first 50 years and beyond
No full textThe Editors of Journal of Applied Crystallography mark the journal's 50th anniversary.</p
Protein crystallography from the perspective of technology developments
No full textEarly on, crystallography was a domain of mineralogy and mathematics and dealt mostly with symmetry properties and imaginary crystal lattices. This changed when Wilhelm Conrad Rontgen discovered X-rays in 1895, and in 1912, Max von Laue and his associates discovered that X-ray irradiated salt crystals would produce diffraction patterns that could reveal the internal atomic periodicity of the crystals. In the same year, the father-and-son team, Henry and Lawrence Bragg successfully solved the first crystal structure of sodium chloride and the era of modern crystallography began. Protein crystallography (PX) started some 20 years later with the pioneering work of British crystallographers. In the past 50-60 years, the achievements of modern crystallography and particularly those in PX have been due to breakthroughs in theoretical and technical advancements such as phasing and direct methods; to more powerful X-ray sources such as synchrotron radiation; to more sensitive and efficient X-ray detectors; to ever faster computers and to improvements in software. The exponential development of PX has been accelerated by the invention and applications of recombinant DNA technology that can yield nearly any protein of interest in large amounts and with relative ease. Novel methods, informatics platforms and technologies for automation and high-throughput have allowed the development of large-scale, high-efficiency macromolecular crystallography efforts in the field of structural genomics. Very recently, the X-ray free-electron laser sources and its applications in PX have shown great potential for revolutionizing the whole field again in the near future
Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches
Get PDFAptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.
emmo-repo/domain-crystallography: v0.1.0
No full textWhat's Changed
Add codeowners for Concept_Templates by @CasperWA in https://github.com/emmo-repo/domain-crystallography/pull/10
Removed equivalence between cif direct part relations and EMMO hasSpatialDirectPart by @jesper-friis in https://github.com/emmo-repo/domain-crystallography/pull/2
Add sample representation concept by @rartino in https://github.com/emmo-repo/domain-crystallography/pull/15
New Contributors
@CasperWA made their first contribution in https://github.com/emmo-repo/domain-crystallography/pull/10
@jesper-friis made their first contribution in https://github.com/emmo-repo/domain-crystallography/pull/2
@rartino made their first contribution in https://github.com/emmo-repo/domain-crystallography/pull/15
Full Changelog: https://github.com/emmo-repo/domain-crystallography/commits/v0.1.
A simple adaptation to a protein crystallography station to facilitate difference X-ray scattering studies
No full textThe X-ray crystallography station I911-2 at MAXLab II (Lund, Sweden) has been adapted to enable difference small- and wide-angle X-ray scattering (SAXS/WAXS) data to be recorded. Modifications to the beamline included a customized flow cell, a motorized flow cell holder, a helium cone, a beam stop, a sample stage and a sample delivery system. This setup incorporated external devices such as infrared lasers, LEDs and reaction mixers to induce conformational changes in macromolecules. This platform was evaluated through proof-of-principle experiments capturing light-induced conformational changes in phytochromes. A difference WAXS signature of conformational changes in a plant aquaporin was also demonstrated using caged calcium
CrystalWalk record at International Union of Crystallography (IUCR)'s crystallographic software database
No full textCrystalWalk record at International Union of Crystallography (IUCR)’s crystallographic software database
Original URL available at https://www.iucr.org/resources/other-directories/software/crystalwalk
Mirrors here and here</p
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