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1
Stewart, P. L., S. D. Fuller, and R. M. Burnett. "Bridging the resolution gap between x-ray crystallography and electron microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 52 (1994): 92–93. http://dx.doi.org/10.1017/s0424820100168190.
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While x-ray crystallography provides atomic resolution structures of proteins and small viruses, electron microscopy can provide complementary structural information on larger assemblies. A significant computational challenge is faced in bridging the resolution gap between the two techniques. X-ray crystallographic data is collected in the range of 2-10 Å, while image reconstructions from electron micrographs are at a resolution of 25-35 Å. A further problem is that density derived from cryo-electron micrographs is distorted by the contrast transfer function of the microscope, whichaccentuates certain resolution bands.A novel combination of electron microscopy and x-ray crystallography has revealed the various structural components forming the capsid of human type 2 adenovirus. An image reconstruction of the intact virus (Fig. 1), derived from cryo-electron micrographs, was deconvolved with an approximate contrast transfer function to mitigate microscope distortions (Fig. 2). A model capsid was calculated from 240 copies of the crystallographic structure of the major capsid protein and filtered to the correct resolution (Fig. 3).
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Blakeley, Matthew. "Macromolecular crystallography using neutrons." Biochemist 36, no. 3 (June 1, 2014): 40–42. http://dx.doi.org/10.1042/bio03603040.
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When you think about macromolecular crystallography, the technique that most often comes to mind is X-ray diffraction and it's no wonder. Over 88000 structures of biological macromolecules – from proteins and nucleic acids to viruses and macromolecular assemblies – have been determined using X-rays, and these have contributed significantly to our understanding of a vast array of biological systems and processes.
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Kojić-Prodić, Biserka. "A century of X-ray crystallography and 2014 international year of X-ray crystallography." Macedonian Journal of Chemistry and Chemical Engineering 34, no. 1 (June 2, 2015): 19. http://dx.doi.org/10.20450/mjcce.2015.663.
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The 100<sup>th</sup> anniversary of the Nobel prize awarded to Max von Laue in 1914 for his discovery of diffraction of X-rays on a crystal marked the beginning of a new branch of science - X-ray crystallography. The experimental evidence of von Laue's discovery was given by physicists W. Friedrich and P. Knipping in 1912. In the same year W. L. Bragg described the analogy between X-rays and visible light and formulated the Bragg's law, a fundamental relation, that connected the wave nature of X-rays and fine structure of a crystal at atomic level. In 1913 the first simple diffractometer was constructed and structure determination started by the Braggs, father and son. In 1915 their discoveries were awarded by Nobel prize in physics. Since then, X-ray diffraction has been basic method for determination of three-dimensional structures of synthetic and natural compounds. The three-dimensional structure of molecule defines its physical, chemical, and biological properties. All over the past century significance of X-ray crystallography has been recognized by about forty Nobel prizes. The examples of X-ray structure analysis, of simple crystals of rock salt, diamond and graphite, and then of complex biomolecules such as B12-vitamin, penicillin, haemoglobin/myoglobin, DNA, and biomolecular complexes such as viruses, chromatin, ribozyme, and other molecular machines, have illustrated the development of the method. Among these big discoveries double helix DNA structure is epochal one of 20<sup>th</sup> century. These discoveries together with many others within X-ray crystallography completely changed our views and helped to be developed different new fields of science such as molecular genetics, biophysics, structural molecular biology, material science, and many others. During the last decade, an implementation of free electron X-ray lasers, a new experimental tool, has opened up femtosecond dynamic crystallography. This highly advanced methodology enables to solve the structures and dynamics of the most complex biological assemblies involved in a cell metabolism. The advancements of science and technology over 20<sup>th</sup> and 21<sup>st</sup>centuries are of great influence on our views in almost all human activities. The importance of X-ray crystallography for science and technology advocates for its high impact on a wide area of research and declares it as highly interdisciplinary science. Briefly saying, crystallography defines the shape of our modern world.<p>The essay is far from being complete and it is concentrated on single crystal diffraction. The wide area of X-ray crystallography hardly can be reviewed in a single article. However, it highlights the most striking examples illustrating some of the milestones over past century.</p><p> </p>
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Humphrey, Charles D., Betty H. Robertson, and B. Khanna. "Hepatitis A Virus Aggregation in Suspensions of Purified Virus." Proceedings, annual meeting, Electron Microscopy Society of America 48, no. 1 (August 12, 1990): 280–81. http://dx.doi.org/10.1017/s042482010018015x.
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Virus capsid structure as determined by x-ray crystallography or by elec imaging of virus particles in thefrozen hydrated state has received con interest during the past decade. Specific functional proteins were localized recently on the Rotavirus capsid by using cryo-electron microscopy, indicating the potential for morphological studies that rel directly to capsid protein function.Most cryo-electron microscopy studies have involved investigation of vir were 40 nm or greater in diameter. Smaller viruses may provide a greate practical difficulty in their preparation and imaging.Highly purified virus for study by x-ray crystallographic studies or ele microscopic evaluation of frozen hydrated particles requires stringent c particle aggregation. Preparations for x-ray crystallography should agg an orderly manner to form arrays and ordered crystals of sufficient size Preparations for cryo-electron microscopy are more desirable when the pa numerous but not over-lapping (Fig. 1). We have experienced uncontrolla aggregation including considerable over-lapping of hepatitis A virus (HA particles when purified preparations were stored in phosphate or tris buffers (Fig. 2).
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Verdaguer, Nuria, Diego Ferrero, and Mathur R. N. Murthy. "Viruses and viral proteins." IUCrJ 1, no. 6 (October 14, 2014): 492–504. http://dx.doi.org/10.1107/s205225251402003x.
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For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.
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Harrison, Stephen. "Virus crystallography from its beginnings until 1978." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C932. http://dx.doi.org/10.1107/s2053273314090676.
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Viruses, first crystallized in the 1930's, were for many years the only exemplars of large macromolecular assemblies accessible to x-ray crystallography. The talk will outline the most interesting biological questions these structures posed, review some of the early technical challenges, and describe some of the specific history that led to the first published virus crystal structure in 1978.
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Burkhardt, Anja, Martin Warmer, Nicolas Stuebe, Jan Roever, Bernd Reime, Saravanan Panneerselvam, Tim Pakendorf, Jan Meyer, Pontus Fischer, and Alke Meents. "X-ray Crystallography at Beamline P11 at PETRA III." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1725. http://dx.doi.org/10.1107/s2053273314082746.
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The bio-imaging and diffraction beamline P11 at PETRA III is dedicated to structure determination of periodic (crystalline) and aperiodic biological samples. The beamline features two experimental endstations: an X-ray microscope and a crystallography experiment. Basis of design was to provide an extremely stable and flexible setup ideally suited for micro and nano beam applications. The X-ray optics consist of a HHL double crystal monochromator, followed by two horizontal deflecting and one vertical deflecting X-ray mirrors. All mirrors are dynamically bendable and used to generate an intermediate focus at 65.5 m from the source with a size of 37 × 221 µm2FWHM (v × h). All experiments are installed on an 8 m long granite support which provides a very stable setup for micro beam experiments. The crystallography endstation is located at the end of the granite at 72.9 m from the source. The experiment is equipped with a high precision single axis goniostat with a combined sphere of confusion of less than 100 nm. X-ray energies are tunable between 5.5 and 30 keV. A second focusing bendable KB mirror system can be used for further demagnification of the secondary source. In this way the beam size can be freely adjusted between 4 × 9 µm2and 300 × 300 µm2FWHM (v × h) with 1013ph/s at 12 keV. Smaller beam sizes down to 1 × 1 µm2with more than 2 × 1011ph/s in the focus can be realized by slitting down the secondary source at the cost of flux. The crystallography endstation is equipped with a Pilatus 6M-F detector which allows fast data collection with up to 25 Hz. Due to the very small beam divergence of the X-ray beam P11 is ideally suited to measure large unit cell systems, such as viruses or large molecular complexes. In addition, the beamline is capable of high-throughput crystallography and fast crystal screening. Crystals can be mounted in less than 10 s using an automatic sample changer. The large sample dewar provides space for 368 crystals.
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Tang, Liang, and John E. Johnson. "Structural Biology of Viruses by the Combination of Electron Cryomicroscopy and X-ray Crystallography†." Biochemistry 41, no. 39 (October 2002): 11517–24. http://dx.doi.org/10.1021/bi020170j.
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Mizianty, Marcin J., Xiao Fan, Jing Yan, Eric Chalmers, Christopher Woloschuk, Andrzej Joachimiak, and Lukasz Kurgan. "Covering complete proteomes with X-ray structures: a current snapshot." Acta Crystallographica Section D Biological Crystallography 70, no. 11 (October 23, 2014): 2781–93. http://dx.doi.org/10.1107/s1399004714019427.
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Structural genomics programs have developed and applied structure-determination pipelines to a wide range of protein targets, facilitating the visualization of macromolecular interactions and the understanding of their molecular and biochemical functions. The fundamental question of whether three-dimensional structures of all proteins and all functional annotations can be determined using X-ray crystallography is investigated. A first-of-its-kind large-scale analysis of crystallization propensity for all proteins encoded in 1953 fully sequenced genomes was performed. It is shown that current X-ray crystallographic knowhow combined with homology modeling can provide structures for 25% of modeling families (protein clusters for which structural models can be obtained through homology modeling), with at least one structural model produced for each Gene Ontology functional annotation. The coverage varies between superkingdoms, with 19% for eukaryotes, 35% for bacteria and 49% for archaea, and with those of viruses following the coverage values of their hosts. It is shown that the crystallization propensities of proteomes from the taxonomic superkingdoms are distinct. The use of knowledge-based target selection is shown to substantially increase the ability to produce X-ray structures. It is demonstrated that the human proteome has one of the highest attainable coverage values among eukaryotes, and GPCR membrane proteins suitable for X-ray structure determination were determined.
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Rossmann, Michael G. "Structure of viruses: a short history." Quarterly Reviews of Biophysics 46, no. 2 (May 2013): 133–80. http://dx.doi.org/10.1017/s0033583513000012.
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AbstractThis review is a partially personal account of the discovery of virus structure and its implication for virus function. Although I have endeavored to cover all aspects of structural virology and to acknowledge relevant individuals, I know that I have favored taking examples from my own experience in telling this story. I am anxious to apologize to all those who I might have unintentionally offended by omitting their work.The first knowledge of virus structure was a result of Stanley's studies of tobacco mosaic virus (TMV) and the subsequent X-ray fiber diffraction analysis by Bernal and Fankuchen in the 1930s. At about the same time it became apparent that crystals of small RNA plant and animal viruses could diffract X-rays, demonstrating that viruses must have distinct and unique structures. More advances were made in the 1950s with the realization by Watson and Crick that viruses might have icosahedral symmetry. With the improvement of experimental and computational techniques in the 1970s, it became possible to determine the three-dimensional, near-atomic resolution structures of some small icosahedral plant and animal RNA viruses. It was a great surprise that the protecting capsids of the first virus structures to be determined had the same architecture. The capsid proteins of these viruses all had a ‘jelly-roll’ fold and, furthermore, the organization of the capsid protein in the virus were similar, suggesting a common ancestral virus from which many of today's viruses have evolved. By this time a more detailed structure of TMV had also been established, but both the architecture and capsid protein fold were quite different to that of the icosahedral viruses. The small icosahedral RNA virus structures were also informative of how and where cellular receptors, anti-viral compounds, and neutralizing antibodies bound to these viruses. However, larger lipid membrane enveloped viruses did not form sufficiently ordered crystals to obtain good X-ray diffraction. Starting in the 1990s, these enveloped viruses were studied by combining cryo-electron microscopy of the whole virus with X-ray crystallography of their protein components. These structures gave information on virus assembly, virus neutralization by antibodies, and virus fusion with and entry into the host cell. The same techniques were also employed in the study of complex bacteriophages that were too large to crystallize. Nevertheless, there still remained many pleomorphic, highly pathogenic viruses that lacked the icosahedral symmetry and homogeneity that had made the earlier structural investigations possible. Currently some of these viruses are starting to be studied by combining X-ray crystallography with cryo-electron tomography.
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Pous, Joan, Christophe Chevalier, Malika Ouldali, Jorge Navaza, Bernard Delmas, and Jean Lepault. "Structure of birnavirus-like particles determined by combined electron cryomicroscopy and X-ray crystallography." Journal of General Virology 86, no. 8 (August 1, 2005): 2339–46. http://dx.doi.org/10.1099/vir.0.80942-0.
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Birnaviruses possess a capsid with a single protein layer in contrast to most double-stranded RNA viruses infecting multicellular eukaryotes. Using freeze-drying and heavy metal shadowing, the capsids of two birnaviruses, infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus, as well as of an IBDV virus-like particle (VLP) are shown to follow the same T=13 laevo icosahedral geometry. The structure of the VLP was determined at a resolution of approximately 15 Å (1·5 nm) by a combination of electron cryomicroscopy and a recently developed three-dimensional reconstruction method, where the scattering density is expressed in terms of symmetry-adapted functions. This reconstruction methodology is well adapted to the icosahedral symmetry of viruses and only requires a small number of images to analyse. The atomic model of the external capsid protein, VP2, recently determined by X-ray crystallography, fits well into the VLP reconstruction and occupies all the electron densities present in the map. Thus, similarly to the IBDV virion, only VP2 forms the icosahedral layer of the VLP. The other components of both VLP and IBDV particles that play a crucial role in the capsid assembly, VP1, VP3 and the peptides arising from the processing of pVP2, do not follow the icosahedral symmetry, allowing them to be involved in other processes such as RNA packaging.
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Duke, E. M. H., and L. N. Johnson. "Macromolecular crystallography at synchrotron radiation sources: current status and future developments." Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences 466, no. 2124 (October 6, 2010): 3421–52. http://dx.doi.org/10.1098/rspa.2010.0448.
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X-ray diffraction with synchrotron radiation (SR) has revealed the atomic structures of numerous biological macromolecules including proteins and protein complexes, nucleic acids and their protein complexes, viruses, membrane proteins and drug targets. The bright SR X-ray beam with its small divergence has made the study of weakly diffracting crystals of large biological molecules possible. The ability to tune the wavelength of the SR beam to the absorption edge of certain elements has allowed anomalous scattering to be exploited for phase determination. We review the developments at synchrotron sources and beamlines from the early days to the present time, and discuss the significance of the results in providing a deeper understanding of the biological function, the design of new therapeutic molecules and time-resolved studies of dynamic events using pump–probe techniques. Radiation damage, a problem with bright X-ray sources, has been partially alleviated by collecting data at low temperature (100 K) but work is ongoing. In the most recent development, free electron laser sources can offer a peak brightness of hard X-rays approximately 10 8 times brighter than that achieved at SR sources. We describe briefly how early experiments at FLASH and Linear Coherent Light Source have shown exciting possibilities for the future.
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Munawar, Ali, Steven Beelen, Ahmad Munawar, Eveline Lescrinier, and Sergei Strelkov. "Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening." International Journal of Molecular Sciences 19, no. 11 (November 20, 2018): 3664. http://dx.doi.org/10.3390/ijms19113664.
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The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015–2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (KD) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.
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Stone, John E., Ryan McGreevy, Barry Isralewitz, and Klaus Schulten. "GPU-accelerated analysis and visualization of large structures solved by molecular dynamics flexible fitting." Faraday Discuss. 169 (2014): 265–83. http://dx.doi.org/10.1039/c4fd00005f.
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Hybrid structure fitting methods combine data from cryo-electron microscopy and X-ray crystallography with molecular dynamics simulations for the determination of all-atom structures of large biomolecular complexes. Evaluating the quality-of-fit obtained from hybrid fitting is computationally demanding, particularly in the context of a multiplicity of structural conformations that must be evaluated. Existing tools for quality-of-fit analysis and visualization have previously targeted small structures and are too slow to be used interactively for large biomolecular complexes of particular interest today such as viruses or for long molecular dynamics trajectories as they arise in protein folding. We present new data-parallel and GPU-accelerated algorithms for rapid interactive computation of quality-of-fit metrics linking all-atom structures and molecular dynamics trajectories to experimentally-determined density maps obtained from cryo-electron microscopy or X-ray crystallography. We evaluate the performance and accuracy of the new quality-of-fit analysis algorithmsvis-à-visexisting tools, examine algorithm performance on GPU-accelerated desktop workstations and supercomputers, and describe new visualization techniques for results of hybrid structure fitting methods.
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Hochstein, Rebecca, Daniel Bollschweiler, Sanjay Dharmavaram, Nathanael G. Lintner, Jürgen M. Plitzko, Robijn Bruinsma, Harald Engelhardt, Mark J. Young, William S. Klug, and C. Martin Lawrence. "Structural studies of Acidianus tailed spindle virus reveal a structural paradigm used in the assembly of spindle-shaped viruses." Proceedings of the National Academy of Sciences 115, no. 9 (February 12, 2018): 2120–25. http://dx.doi.org/10.1073/pnas.1719180115.
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The spindle-shaped virion morphology is common among archaeal viruses, where it is a defining characteristic of many viral families. However, structural heterogeneity intrinsic to spindle-shaped viruses has seriously hindered efforts to elucidate the molecular architecture of these lemon-shaped capsids. We have utilized a combination of cryo-electron microscopy and X-ray crystallography to study Acidianus tailed spindle virus (ATSV). These studies reveal the architectural principles that underlie assembly of a spindle-shaped virus. Cryo-electron tomography shows a smooth transition from the spindle-shaped capsid into the tubular-shaped tail and allows low-resolution structural modeling of individual virions. Remarkably, higher-dose 2D micrographs reveal a helical surface lattice in the spindle-shaped capsid. Consistent with this, crystallographic studies of the major capsid protein reveal a decorated four-helix bundle that packs within the crystal to form a four-start helical assembly with structural similarity to the tube-shaped tail structure of ATSV and other tailed, spindle-shaped viruses. Combined, this suggests that the spindle-shaped morphology of the ATSV capsid is formed by a multistart helical assembly with a smoothly varying radius and allows construction of a pseudoatomic model for the lemon-shaped capsid that extends into a tubular tail. The potential advantages that this novel architecture conveys to the life cycle of spindle-shaped viruses, including a role in DNA ejection, are discussed.
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Benton, Donald J., Andrea Nans, Lesley J. Calder, Jack Turner, Ursula Neu, Yi Pu Lin, Esther Ketelaars, et al. "Influenza hemagglutinin membrane anchor." Proceedings of the National Academy of Sciences 115, no. 40 (September 17, 2018): 10112–17. http://dx.doi.org/10.1073/pnas.1810927115.
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Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the “membrane anchor” portion of the molecule. Since this region has a role in membrane fusion, we have determined its structure by analyzing the intact, full-length molecule in a detergent micelle, using cryo-EM. We have also compared the structures of full-length HA−detergent micelles with full-length HA−Fab complex detergent micelles, to describe an infectivity-neutralizing monoclonal Fab that binds near the ectodomain membrane anchor junction. We determine a high-resolution HA structure which compares favorably in detail with the structure of the ectodomain seen by X-ray crystallography; we detect, clearly, all five carbohydrate side chains of HA; and we find that the ectodomain is joined to the membrane anchor by flexible, eight-residue-long, linkers. The linkers extend into the detergent micelle to join a central triple-helical structure that is a major component of the membrane anchor.
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Crowther, R. A., and K. C. Holmes. "John Thomas Finch. 28 February 1930—5 December 2017." Biographical Memoirs of Fellows of the Royal Society 66 (December 19, 2018): 183–99. http://dx.doi.org/10.1098/rsbm.2018.0028.
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John Finch was a gifted experimentalist who used X-ray crystallography and electron microscopy to elucidate the structures of important biological assemblies, particularly viruses and chromatin. When he started research in the 1950s, little was known about the structure of viruses, and the methods for investigating them were fairly limited. His early work on crystals of viruses was important in establishing their symmetry, and later with the electron microscope he mapped out the molecular structure of many virus coats. His observations on negatively stained preparations demonstrated that images of particles prepared in this way represented projections of fully stained embedded particles, not merely one-sided footprints. This was very relevant to the development of methods for making three-dimensional maps of specimens from electron micrographs. Later, besides further studies of viruses, he worked on many other systems, including chromatin, nucleosomes and tRNA. John was very much a team player and held an important place as the key experimentalist in many influential collaborations, investigating a diverse range of biological specimens.
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Hejny, Clivia, and Vasily S. Minkov. "High-pressure crystallography of periodic and aperiodic crystals." IUCrJ 2, no. 2 (March 1, 2015): 218–29. http://dx.doi.org/10.1107/s2052252514025482.
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More than five decades have passed since the first single-crystal X-ray diffraction experiments at high pressure were performed. These studies were applied historically to geochemical processes occurring in the Earth and other planets, but high-pressure crystallography has spread across different fields of science including chemistry, physics, biology, materials science and pharmacy. With each passing year, high-pressure studies have become more precise and comprehensive because of the development of instrumentation and software, and the systems investigated have also become more complicated. Starting with crystals of simple minerals and inorganic compounds, the interests of researchers have shifted to complicated metal–organic frameworks, aperiodic crystals and quasicrystals, molecular crystals, and even proteins and viruses. Inspired by contributions to the microsymposium `High-Pressure Crystallography of Periodic and Aperiodic Crystals' presented at the 23rd IUCr Congress and General Assembly, the authors have tried to summarize certain recent results of single-crystal studies of molecular and aperiodic structures under high pressure. While the selected contributions do not cover the whole spectrum of high-pressure research, they demonstrate the broad diversity of novel and fascinating results and may awaken the reader's interest in this topic.
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Šašková, Klára Grantz, Milan Kožíšek, Pavlína Řezáčová, Jiří Brynda, Tatyana Yashina, Ron M. Kagan, and Jan Konvalinka. "Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir." Journal of Virology 83, no. 17 (June 17, 2009): 8810–18. http://dx.doi.org/10.1128/jvi.00451-09.
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ABSTRACT Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2′ binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.
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Bauer, Paul H., Cunqi Cui, Thilo Stehle, Stephen C. Harrison, James A. DeCaprio, and Thomas L. Benjamin. "Discrimination between Sialic Acid-Containing Receptors and Pseudoreceptors Regulates Polyomavirus Spread in the Mouse." Journal of Virology 73, no. 7 (July 1, 1999): 5826–32. http://dx.doi.org/10.1128/jvi.73.7.5826-5832.1999.
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ABSTRACT Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal α-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.
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Li, Na, Zhiqiang Li, Yan Fu, and Sheng Cao. "Cryo-EM Studies of Virus-Antibody Immune Complexes." Virologica Sinica 35, no. 1 (January 8, 2020): 1–13. http://dx.doi.org/10.1007/s12250-019-00190-5.
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AbstractAntibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design. Cryo-electron microscopy (cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3 (picornaviruses) and T = 3 (flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.
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Xiao, Chuan, Tobias J. Tuthill, Carol M. Bator Kelly, Lisa J. Challinor, Paul R. Chipman, Richard A. Killington, David J. Rowlands, Alister Craig, and Michael G. Rossmann. "Discrimination among Rhinovirus Serotypes for a Variant ICAM-1 Receptor Molecule." Journal of Virology 78, no. 18 (September 15, 2004): 10034–44. http://dx.doi.org/10.1128/jvi.78.18.10034-10044.2004.
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ABSTRACT Intercellular adhesion molecule 1 (ICAM-1) is the cellular receptor for the major group of human rhinovirus serotypes, including human rhinovirus 14 (HRV14) and HRV16. A naturally occurring variant of ICAM-1, ICAM-1Kilifi, has altered binding characteristics with respect to different HRV serotypes. HRV14 binds to ICAM-1 only transiently at physiological temperatures but forms a stable complex with ICAM-1Kilifi. Conversely, HRV16 forms a stable complex with ICAM-1 but does not bind to ICAM-1Kilifi. The three-dimensional structures of HRV14 and HRV16, complexed with ICAM-1, and the structure of HRV14, complexed with ICAM-1Kilifi, have been determined by cryoelectron microscopy (cryoEM) image reconstruction to a resolution of approximately 10 Å. Structures determined by X-ray crystallography of both viruses and of ICAM-1 were fitted into the cryoEM density maps. The interfaces between the viruses and receptors contain extensive ionic networks. However, the interactions between the viruses and ICAM-1Kilifi contain one less salt bridge than between the viruses and ICAM-1. As HRV16 has fewer overall interactions with ICAM-1 than HRV14, the absence of this charge interaction has a greater impact on the binding of ICAM-1Kilifi to HRV16 than to HRV14.
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Jüstel, Dominik, Gero Friesecke, and Richard James. "Beyond Crystals and Plane Waves - Generalizing the von Laue Condition." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C524. http://dx.doi.org/10.1107/s2053273314094753.
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The success of X-ray crystallography is due to the relationship between the symmetries of plane wave radiation and crystal structure. The resulting sharply peaked diffraction patterns allow to reconstruct translational symmetry information directly via the von Laue condition. A natural question to ask is whether one can design radiation that is in a similar way related to certain non-crystalline structures such as the so-called 'objective structures' whose symmetries were analyzed recently in [1]. Ideas and results surrounding these questions are discussed. It is shown that the use of novel forms of radiation might have the potential to improve the structural analysis of structures such as nanotubes, fullerenes, protein substructures or tails and capsids of certain viruses, which are non-crystalline but highly symmetric.
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Sanna, Giuseppina, Silvia Madeddu, Gabriele Giliberti, Sandra Piras, Marta Struga, Małgorzata Wrzosek, Grażyna Kubiak-Tomaszewska, et al. "Synthesis and Biological Evaluation of Novel Indole-Derived Thioureas." Molecules 23, no. 10 (October 7, 2018): 2554. http://dx.doi.org/10.3390/molecules23102554.
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A series of 2-(1H-indol-3-yl)ethylthiourea derivatives were prepared by condensation of 2-(1H-indol-3-yl)ethanamine with appropriate aryl/alkylisothiocyanates in anhydrous media. The structures of the newly synthesized compounds were confirmed by spectroscopic analysis and the molecular structures of 8 and 28 were confirmed by X-ray crystallography. All obtained compounds were tested for antimicrobial activity against Gram-positive cocci, Gram-negative rods and for antifungal activity. Microbiological evaluation was carried out over 20 standard strains and 30 hospital strains. Compound 6 showed significant inhibition against Gram-positive cocci and had inhibitory effect on the S. aureus topoisomerase IV decatenation activity and S. aureus DNA gyrase supercoiling activity. Compounds were tested for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses, including HIV-1 and other several important human pathogens. Interestingly, derivative 8 showed potent activity against HIV-1 wild type and variants bearing clinically relevant mutations. Newly synthesized tryptamine derivatives showed also a wide spectrum activity, proving to be active against positive- and negative-sense RNA viruses.
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Crowther, R. A. "Sir Aaron Klug OM. 11 August 1926—20 November 2018." Biographical Memoirs of Fellows of the Royal Society 68 (January 8, 2020): 273–96. http://dx.doi.org/10.1098/rsbm.2019.0034.
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Aaron Klug made outstanding contributions to the development of structural molecular biology. An early interest in viruses, stemming from work with Rosalind Franklin, prompted him to think deeply about extracting the information contained in electron micrographs. As a result, he proposed a method for making three-dimensional maps of biological specimens from the projected images given by micrographs. For this development and its application to complex molecular assemblies, he was awarded the 1982 Nobel Prize in Chemistry. The recent revolution in biological structure determination, whereby atomic structures can now be determined from micrographs of frozen hydrated specimens, derives from this initial breakthrough. With colleagues, Aaron applied X-ray crystallography and electron microscopy to determine the structures and thereby understand the functions of many biological assemblies, including viruses, transfer RNA, chromatin and zinc fingers. He also made important forays into the pathogenesis of Alzheimer's disease and related dementias. Aaron was director of the MRC Laboratory of Molecular Biology in Cambridge from 1986 to 1996 and President of the Royal Society from 1995 to 2000.
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Lévêque, Vincent J. P., Robert B. Johnson, Stephen Parsons, Jianxin Ren, Congping Xie, Faming Zhang, and Q. May Wang. "Identification of a C-Terminal Regulatory Motif in Hepatitis C Virus RNA-Dependent RNA Polymerase: Structural and Biochemical Analysis." Journal of Virology 77, no. 16 (August 15, 2003): 9020–28. http://dx.doi.org/10.1128/jvi.77.16.9020-9028.2003.
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ABSTRACT The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-Å resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to ∼50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.
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Li, Jiao, Yujia Wang, Xiaomeng Hao, Shasha Li, Jia Jia, Yan Guan, Zonggen Peng, et al. "Broad-Spectrum Antiviral Natural Products from the Marine-Derived Penicillium sp. IMB17-046." Molecules 24, no. 15 (August 2, 2019): 2821. http://dx.doi.org/10.3390/molecules24152821.
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A new pyrazine derivative, trypilepyrazinol (1), a new α-pyrone polyketide, (+)-neocitreoviridin (2), and a new ergostane analogue, 3β-hydroxyergosta-8,14,24(28)-trien-7-one (3), were isolated and characterized along with five known compounds from the marine-derived fungus Penicillium sp. IMB17-046. The structures of these new compounds were determined using spectroscopic data analyses (HRESIMS, 1D- and 2D-NMR), X-ray crystallography analysis, and TDDFT ECD calculation. Compounds 1 and 3 exhibited broad-spectrum antiviral activities against different types of viruses, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza A virus (IAV), with IC50 values ranging from 0.5 to 7.7 μM. Compounds 1 and 2 showed antibacterial activities against Helicobacter pylori, a causative pathogen of various gastric diseases, with minimum inhibitory concentration (MIC) values of 1–16 μg/mL.
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Blaum, Bärbel S., Ursula Neu, Thomas Peters, and Thilo Stehle. "Spin ballet for sweet encounters: saturation-transfer difference NMR and X-ray crystallography complement each other in the elucidation of protein–glycan interactions." Acta Crystallographica Section F Structural Biology Communications 74, no. 8 (July 26, 2018): 451–62. http://dx.doi.org/10.1107/s2053230x18006581.
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Biomolecular NMR spectroscopy has limitations in the determination of protein structures: an inherent size limit and the requirement for expensive and potentially difficult isotope labelling pose considerable hurdles. Therefore, structural analysis of larger proteins is almost exclusively performed by crystallography. However, the diversity of biological NMR applications outperforms that of any other structural biology technique. For the characterization of transient complexes formed by proteins and small ligands, notably oligosaccharides, one NMR technique has recently proven to be particularly powerful: saturation-transfer difference NMR (STD-NMR) spectroscopy. STD-NMR experiments are fast and simple to set up, with no general protein size limit and no requirement for isotope labelling. The method performs best in the moderate-to-low affinity range that is of interest in most of glycobiology. With small amounts of unlabelled protein, STD-NMR experiments can identify hits from mixtures of potential ligands, characterize mutant proteins and pinpoint binding epitopes on the ligand side. STD-NMR can thus be employed to complement and improve protein–ligand complex models obtained by other structural biology techniques or by purely computational means. With a set of protein–glycan interactions from our own work, this review provides an introduction to the technique for structural biologists. It exemplifies how crystallography and STD-NMR can be combined to elucidate protein–glycan (and other protein–ligand) interactions in atomic detail, and how the technique can extend structural biology from simplified systems amenable to crystallization to more complex biological entities such as membranes, live viruses or entire cells.
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Bennett, Antonette, Robert McKenna, and Mavis Agbandje-McKenna. "A Comparative Analysis of the Structural Architecture of ssDNA Viruses." Computational and Mathematical Methods in Medicine 9, no. 34 (2008): 183–96. http://dx.doi.org/10.1080/17486700802168247.
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Virus assembly, utilizing a limited number of viral coat protein (CP or VP) building blocks, is an excellent example of a directed macromolecular interaction occurring in nature. Two basic principles govern the assembly of spherical (icosahedral) viruses: (i) Genetic Economy – the encapsidated genome encodes a single or few CPs that assemble a protective shell (the viral capsid); and (ii) specificity – the CPs have to fold to recognize each other and form exact CP–CP interfacial interactions during the assembly pathway. Using a variety of biophysical techniques, including X-ray crystallography and cryo-electron microscopy, combined with homology model building, biochemistry and molecular biology, the nature of the interactions between protein–protein subunits and protein–nucleic acid that facilitate viral capsid assembly have been studied. This review discusses both the similarities and differences that have been elucidated for ssDNAMicroviridae, Geminiviridae, and Parvoviridaevirus families. TheMicroviridaerepresent a family of bacteriophages that utilize several CPs and scaffold proteins to assemble aT= 1 icosahedral capsid, theGeminiviridaeplant viruses assemble a unique twinned quasi-isometric (geminate) pseudoT= 1 virion assembled from a single CP and theParvoviridaerepresent animal viruses whoseT= 1 capsids are formed from the common overlapping region of two to four VPs that have unique N-terminal extensions. A survey of the three-dimensional (3D) data available for these viruses shows that they utilize structural commonalities, facilitated by disparate CP/VP amino acid sequences for the successful assembly of mature infectious virions.
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Garman, Elspeth F. "Antiviral adhesion molecular mechanisms for influenza: W. G. Laver's lifetime obsession." Philosophical Transactions of the Royal Society B: Biological Sciences 370, no. 1661 (February 5, 2015): 20140034. http://dx.doi.org/10.1098/rstb.2014.0034.
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Infection by the influenza virus depends firstly on cell adhesion via the sialic-acid-binding viral surface protein, haemagglutinin, and secondly on the successful escape of progeny viruses from the host cell to enable the virus to spread to other cells. To achieve the latter, influenza uses another glycoprotein, the enzyme neuraminidase (NA), to cleave the sialic acid receptors from the surface of the original host cell. This paper traces the development of anti-influenza drugs, from the initial suggestion by MacFarlane Burnet in 1948 that an effective ‘competitive poison’ of the virus' NA might be useful in controlling infection by the virus, through to the determination of the structure of NA by X-ray crystallography and the realization of Burnet's idea with the design of NA inhibitors. A focus is the contribution of the late William Graeme Laver, FRS, to this research.
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Afione, Sandra, Michael A. DiMattia, Sujata Halder, Giovanni Di Pasquale, Mavis Agbandje-McKenna, and John A. Chiorini. "Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region." Journal of Virology 89, no. 3 (November 19, 2014): 1660–72. http://dx.doi.org/10.1128/jvi.02503-14.
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ABSTRACTAs a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid (SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the βHI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropismin vivoas well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus.IMPORTANCEDespite the fact that a majority of AAVs utilize sialic acid for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and studied using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid-dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropismin vivoas well as resistance to neutralization by antibodies raised against the wild-type virus.
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Barker, Adam I., Robert J. Southworth-Davies, Karthik S. Paithankar, Ian Carmichael, and Elspeth F. Garman. "Room-temperature scavengers for macromolecular crystallography: increased lifetimes and modified dose dependence of the intensity decay." Journal of Synchrotron Radiation 16, no. 2 (February 25, 2009): 205–16. http://dx.doi.org/10.1107/s0909049509003343.
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The advent of highly intense wiggler and undulator beamlines has reintroduced the problem of X-ray radiation damage in protein crystals even at cryogenic temperatures (100 K). Although cryocrystallography can be utilized for the majority of protein crystals, certain macromolecular crystals (e.g. of viruses) suffer large increases in mosaicity upon flash cooling and data are still collected at room temperature (293 K). An alternative mechanism to cryocooling for prolonging crystal lifetime is the use of radioprotectants. These compounds are able to scavenge the free radical species formed upon X-ray irradiation which are thought to be responsible for part of the observed damage. Three putative radioprotectants, ascorbate, 1,4-benzoquinone and 2,2,6,6-tetramethyl-4-piperidone (TEMP), were tested for their ability to prolong lysozyme crystal lifetimes at 293 K. Plots of relative summed intensity against dose were used as a metric to assess radioprotectant ability: ascorbate and 1,4-benzoquinone appear to be effective, whereas studies on TEMP were inconclusive. Ascorbate, which scavenges OH^{\bullet} radicals (k OH = 8 × 109 M −1 s−1) and electrons with a lower rate constant (k e-(aq) = 3.0 × 108 M −1 s−1), doubled the crystal dose tolerance, whereas 1,4-benzoquinone, which also scavenges both OH^{\bullet} radicals (k OH = 1.2 × 109 M −1 s−1) and electrons (k e-(aq) = 1.2 × 1010 M −1 s−1), offered a ninefold increase in dose tolerance at the dose rates used. Pivotally, these preliminary results on a limited number of samples show that the two scavengers also induced a striking change in the dose dependence of the intensity decay from a first-order to a zeroth-order process.
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Spence, John. "Research at the NSF STC for Biology with X-ray lasers." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C296. http://dx.doi.org/10.1107/s2053273314097034.
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"The NSF BioXFEL Science and Technology Center (STC) is a new consortium of six research campuses devoted to the application of x-ray free-electron lasers (XFELs) to structural biology. Over the last four years a variety of approaches have been made to the observation of protein structure and dynamics for various classes of proteins. The Linac Coherent Light source at SLAC, the first hard-Xray EXFEL, provides intense coherent hard X-ray pulses at 120 Hz which vaporize protein when focussed to a sub-micron beam. Atomic-resolution Bragg diffraction patterns are nevertheless obtained using 50 fs pulses prior to the onset of significant damage, in this ""diffract-then-destroy"" mode, which outruns radiation damage. This use of short pulses instead of freezing samples to reduce radiation damage therefore opens the way to the study of protein dynamics at room temperature in a native environment. I'll review the work of several groups using a range of approaches to different types of sample, including the following: 1. Differences between the frozen sychrotron structure of GPCR proteins and the RT XFEL structure [1]. 2. Pump-probe dynamic structures in Photosynthesis [2]. 3. XFEL study of 2D protein crystals [3]. 4. Prospects for improved resolution in XFEL imaging from single particles such as viruses, where patterns can be obtained from a single virus. 5. New ideas - the Lipid Cubic Phase injector (which allows protein nanocrystals to be studied also at sychrotrons) [4], prospects for fast Laue diffraction using coherent attosecond X-ray lasers, ab-initio phasing [5], the use of angular correlation functions for analysis of fast solution scattering, and two-color opportunites for serial femtosecond crystallography (SFX). See [6] for a recent review of the field. 1. W.Liu et al Science 342, 1521 (2013) 2. A.Aquila et al Optics Express 20, 2706 (2012) 3. M.Frank et al IUCrJ (2014) In press. 4. U.Weierstall et al Nature Comms. (2014) In press. 5. J. Spence et al Optics Express 19, 2866 (2011). 6. J. Spence et al Rep. Prog. Phys. 75, 102601 (2012)."
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Banchenko, Sofia, Ferdinand Krupp, Christine Gotthold, Jörg Bürger, Andrea Graziadei, Francis J. O’Reilly, Ludwig Sinn, et al. "Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses." PLOS Pathogens 17, no. 8 (August 2, 2021): e1009775. http://dx.doi.org/10.1371/journal.ppat.1009775.
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Viruses have evolved means to manipulate the host’s ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins.
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Лунина, Н. Л., and N. L. Lunina. "The Use of Connected Masks for Reconstructing the Single Particle Image from X-Ray Diffraction Data. II. The Dependence of the Accuracy of the Solution on the Sampling Step of Experimental Data." Mathematical Biology and Bioinformatics 10, no. 2 (December 3, 2015): 508–25. http://dx.doi.org/10.17537/2015.10.508.
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Advances in the methodology of the X-ray diffraction experiments leads to a possibility to register the rays scattered by large isolated biological particles (viruses and individual cells) but not only by crystalline samples. The experiment with an isolated particle provides researchers with the intensities of the scattered rays for the continuous spectrum of scattering vectors. Such experiment gives much more experimental data than an experiment with a crystalline sample where the information is limited to a set of Bragg reflections. This opens up additional opportunities in solving underlying problem of X-ray crystallography, namely, calculating phase values for the scattered waves needed to restore the structure of the object under study. In practice, the original continuous diffraction pattern is sampled, reduced to the values at grid points in the space of scattering vectors (in the reciprocal space). The sampling step determines the amount of the information involved in solving the phase problem and the complexity of the necessary calculations. In this paper, we investigate the effect of the sampling step on the accuracy of the phase problem solution obtained by the method proposed earlier by the authors. It is shown that an expected improvement of the accuracy of the solution with the reducing the sampling step continues even after crossing the Nyquist limit defined as the inverse of the double size of the object under study.
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Kumar, Dilip, Xinzhe Yu, Sue E. Crawford, Rodolfo Moreno, Joanita Jakana, Banumathi Sankaran, Ramakrishnan Anish, et al. "2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity." Science Advances 6, no. 16 (April 2020): eaay6410. http://dx.doi.org/10.1126/sciadv.aay6410.
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In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 caps the nascent transcripts synthesized from the genomic dsRNA segments by the RV polymerase VP1 within the particle core. Here, from cryo–electron microscopy, x-ray crystallography, and biochemical analyses, we show that VP3 forms a stable tetrameric assembly with each subunit having a modular domain organization, which uniquely integrates five distinct enzymatic steps required for capping the transcripts. In addition to the previously known guanylyl- and methyltransferase activities, we show that VP3 exhibits hitherto unsuspected RNA triphosphatase activity necessary for initiating transcript capping and RNA helicase activity likely required for separating the RNA duplex formed transiently during endogenous transcription. From our studies, we propose a new mechanism for how VP3 inside the virion core caps the nascent transcripts exiting from the polymerase.
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Xiang, Ye, Ulrich Baxa, Ying Zhang, Alasdair C. Steven, Gentry L. Lewis, James L. Van Etten, and Michael G. Rossmann. "Crystal Structure of a Virus-Encoded Putative Glycosyltransferase." Journal of Virology 84, no. 23 (September 22, 2010): 12265–73. http://dx.doi.org/10.1128/jvi.01303-10.
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ABSTRACT The chloroviruses (family Phycodnaviridae), unlike most viruses, encode some, if not most, of the enzymes involved in the glycosylation of their structural proteins. Annotation of the gene product B736L from chlorovirus NY-2A suggests that it is a glycosyltransferase. The structure of the recombinantly expressed B736L protein was determined by X-ray crystallography to 2.3-Å resolution, and the protein was shown to have two nucleotide-binding folds like other glycosyltransferase type B enzymes. This is the second structure of a chlorovirus-encoded glycosyltransferase and the first structure of a chlorovirus type B enzyme to be determined. B736L is a retaining enzyme and belongs to glycosyltransferase family 4. The donor substrate was identified as GDP-mannose by isothermal titration calorimetry and was shown to bind into the cleft between the two domains in the protein. The active form of the enzyme is probably a dimer in which the active centers are separated by about 40 Å.
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Drayman, Nir, Jennifer K. DeMarco, Krysten A. Jones, Saara-Anne Azizi, Heather M. Froggatt, Kemin Tan, Natalia Ivanovna Maltseva, et al. "Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2." Science 373, no. 6557 (July 20, 2021): 931–36. http://dx.doi.org/10.1126/science.abg5827.
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There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).
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Huang, Lin-Ya, Ami Patel, Robert Ng, Edward Blake Miller, Sujata Halder, Robert McKenna, Aravind Asokan, and Mavis Agbandje-McKenna. "Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site." Journal of Virology 90, no. 11 (March 9, 2016): 5219–30. http://dx.doi.org/10.1128/jvi.00161-16.
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ABSTRACTThe adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants—S472R, V473D, and N500E—escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface “hot spot” dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering.IMPORTANCEThe adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect to transduction efficiency and the detrimental effects of preexisting host antibodies. This study aimed to identify key capsid regions which can be engineered to overcome these limitations. A sialic glycan receptor recognition pocket was identified in AAV1 and its closely related AAV6, using X-ray crystallography. The site was confirmed by mutagenesis followed by cell binding and transduction assays. Significantly, residues controlling gene expression efficiency, as well as antibody escape variants, were also identified. This study thus provides, at the amino acid level, information for rational structural engineering of AAV vectors with improved therapeutic efficacy.
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Prins, Kathleen C., Sebastien Delpeut, Daisy W. Leung, Olivier Reynard, Valentina A. Volchkova, St Patrick Reid, Parameshwaran Ramanan, et al. "Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs." Journal of Virology 84, no. 6 (January 13, 2010): 3004–15. http://dx.doi.org/10.1128/jvi.02459-09.
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ABSTRACT Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-α/β responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-α/β production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.
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Kaufmann, Bärbel, Valorie D. Bowman, Yi Li, Jozsef Szelei, Peter J. Waddell, Peter Tijssen, and Michael G. Rossmann. "Structure of Penaeus stylirostris Densovirus, a Shrimp Pathogen." Journal of Virology 84, no. 21 (August 11, 2010): 11289–96. http://dx.doi.org/10.1128/jvi.01240-10.
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ABSTRACT Penaeus stylirostris densovirus (PstDNV), a pathogen of penaeid shrimp, causes significant damage to farmed and wild shrimp populations. In contrast to other parvoviruses, PstDNV probably has only one type of capsid protein that lacks the phospholipase A2 activity that has been implicated as a requirement during parvoviral host cell infection. The structure of recombinant virus-like particles, composed of 60 copies of the 37.5-kDa coat protein, the smallest parvoviral capsid protein reported thus far, was determined to 2.5-Å resolution by X-ray crystallography. The structure represents the first near-atomic resolution structure within the genus Brevidensovirus. The capsid protein has a β-barrel “jelly roll” motif similar to that found in many icosahedral viruses, including other parvoviruses. The N-terminal portion of the PstDNV coat protein adopts a “domain-swapped” conformation relative to its twofold-related neighbor similar to the insect parvovirus Galleria mellonella densovirus (GmDNV) but in stark contrast to vertebrate parvoviruses. However, most of the surface loops have little structural resemblance to any of the known parvoviral capsid proteins.
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Li, Qingxin, and CongBao Kang. "Insights into Structures and Dynamics of Flavivirus Proteases from NMR Studies." International Journal of Molecular Sciences 21, no. 7 (April 5, 2020): 2527. http://dx.doi.org/10.3390/ijms21072527.
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Nuclear magnetic resonance (NMR) spectroscopy plays important roles in structural biology and drug discovery, as it is a powerful tool to understand protein structures, dynamics, and ligand binding under physiological conditions. The protease of flaviviruses is an attractive target for developing antivirals because it is essential for the maturation of viral proteins. High-resolution structures of the proteases in the absence and presence of ligands/inhibitors were determined using X-ray crystallography, providing structural information for rational drug design. Structural studies suggest that proteases from Dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV) exist in open and closed conformations. Solution NMR studies showed that the closed conformation is predominant in solution and should be utilized in structure-based drug design. Here, we reviewed solution NMR studies of the proteases from these viruses. The accumulated studies demonstrated that NMR spectroscopy provides additional information to understand conformational changes of these proteases in the absence and presence of substrates/inhibitors. In addition, NMR spectroscopy can be used for identifying fragment hits that can be further developed into potent protease inhibitors.
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Cho, Jae-Hyun, Baoyu Zhao, Jie Shi, Nowlan Savage, Qingliang Shen, James Byrnes, Lin Yang, Wonmuk Hwang, and Pingwei Li. "Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses." Proceedings of the National Academy of Sciences 117, no. 12 (March 9, 2020): 6550–58. http://dx.doi.org/10.1073/pnas.1920582117.
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The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85β subunit. Here we present the mechanism underlying the molecular recognition of the p85β subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85β of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85β. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1EDexists in a dynamic equilibrium between p85β-binding–competent and –incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1EDproteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85β. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1EDcan result in the faster hijacking of p85β compared to Ud NS1ED, although the former has a lower affinity to p85β than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.
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Xiong, Xiaoli, Davide Corti, Junfeng Liu, Debora Pinna, Mathilde Foglierini, Lesley J. Calder, Stephen R. Martin, et al. "Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody." Proceedings of the National Academy of Sciences 112, no. 30 (July 13, 2015): 9430–35. http://dx.doi.org/10.1073/pnas.1510816112.
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H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.
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Holý, Antonín, Miloš Buděšínský, Jaroslav Podlaha, and Ivana Císařová. "Synthesis of Quaternary 1-[2-(Phosphonomethoxy)ethyl] Derivatives of 2,4-Diaminopyrimidine and Related Acyclic Nucleotide Analogs." Collection of Czechoslovak Chemical Communications 64, no. 2 (1999): 242–56. http://dx.doi.org/10.1135/cccc19990242.
Full textAbstract:
Quaternization of 2,4-diaminopyrimidine (2) by diisopropyl 2-chloroethoxymethanephosphonate (3) followed by bromotrimethylsilane treatment and subsequent hydrolysis gave zwitterionic N1-[2-(phosphonomethoxy)ethyl] derivative, hydrogen {[2-(2,4-diaminopyrimidin-1-io)ethoxy]methyl}phosphonate (5). Its structure was confirmed by X-ray crystallography. The same product was obtained from 2-amino-4-[(dimethylaminomethylene)amino]pyrimidine (6) by an analogous reaction sequence followed by an aqueous ammonia treatment after the transsilylation reaction. Also the quaternizations of 4,6-diaminopyrimidine (7) and 2,4,6-triaminopyrimidine (8) with the halo derivative 3 afforded the zwitterionic N1-substituted compounds 9 and 10, respectively. In contrast to this regiospecific reaction, 2-aminopyrimidine (11) gave on treatment with compound 3 and following deprotection the exo-N2-isomer 13. This compound was also obtained by the reaction starting from 2-[(dimethylaminomethylene)amino]pyrimidine (12) which was prepared by treatment of compound 11 with dimethylformamide dineopentyl acetal. Also 2,3-diaminopyridine (14) gave by the above reaction a mixture of 2-amino-3-{[2-(phosphonomethoxy)ethyl]amino}pyridine (15) and quaternary N1-[2-(phosphonomethoxy)ethyl] derivative (16). None of these analogs of the antiviral PMEDAP exhibited any antiviral activity against DNA viruses or retroviruses, nor any cytostatic activity.
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Wu, Nicholas C., Meng Yuan, Sandhya Bangaru, Deli Huang, Xueyong Zhu, Chang-Chun D. Lee, Hannah L. Turner, et al. "A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody." PLOS Pathogens 16, no. 12 (December 4, 2020): e1009089. http://dx.doi.org/10.1371/journal.ppat.1009089.
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Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.
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Twu, Karen Y., Rei-Lin Kuo, Jesper Marklund, and Robert M. Krug. "The H5N1 Influenza Virus NS Genes Selected after 1998 Enhance Virus Replication in Mammalian Cells." Journal of Virology 81, no. 15 (May 23, 2007): 8112–21. http://dx.doi.org/10.1128/jvi.00006-07.
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ABSTRACT The NS1A proteins of human influenza A viruses bind CPSF30, a cellular factor required for the processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including beta interferon mRNA. Here we show that the NS1A protein of the pathogenic H5N1 influenza A/Hong Kong/483/97 (HK97) virus isolated from humans has an intrinsic defect in CPSF30 binding. It does not bind CPSF30 in vitro and causes high beta interferon mRNA production and reduced virus replication in MDCK cells when expressed in a recombinant virus in which the other viral proteins are encoded by influenza A/Udorn/72. We traced this defect to the identities of amino acids 103 and 106 in the HK97 NS1A protein, which differ from the consensus amino acids, F and M, respectively, found in the NS1A proteins of almost all human influenza A virus strains. X-ray crystallography has shown that F103 and M106, which are not part of the CPSF30 binding pocket of the NS1A protein, stabilize the NS1A-CPSF30 complex. In contrast to the HK97 NS1A protein, the NS1A proteins of H5N1 viruses isolated from humans after 1998 contain F103 and M106 and hence bind CPSF30 in vitro and do not attenuate virus replication. The HK97 NS1A protein is less attenuating when expressed in a virus that also encodes the other internal HK97 proteins and under these conditions binds to CPSF30 to a substantial extent in vivo. Consequently, these internal HK97 proteins largely compensate for the absence of F103 and M106, presumably by stabilizing the NS1A-CPSF30 complex.
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Georgescauld, Florian, Yuyu Song, and Alain Dautant. "Structure, Folding and Stability of Nucleoside Diphosphate Kinases." International Journal of Molecular Sciences 21, no. 18 (September 16, 2020): 6779. http://dx.doi.org/10.3390/ijms21186779.
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Nucleoside diphosphate kinases (NDPK) are oligomeric proteins involved in the synthesis of nucleoside triphosphates. Their tridimensional structure has been solved by X-ray crystallography and shows that individual subunits present a conserved ferredoxin fold of about 140 residues in prokaryotes, archaea, eukaryotes and viruses. Monomers are functionally independent from each other inside NDPK complexes and the nucleoside kinase catalytic mechanism involves transient phosphorylation of the conserved catalytic histidine. To be active, monomers must assemble into conserved head to tail dimers, which further assemble into hexamers or tetramers. The interfaces between these oligomeric states are very different but, surprisingly, the assembly structure barely affects the catalytic efficiency of the enzyme. While it has been shown that assembly into hexamers induces full formation of the catalytic site and stabilizes the complex, it is unclear why assembly into tetramers is required for function. Several additional activities have been revealed for NDPK, especially in metastasis spreading, cytoskeleton dynamics, DNA binding and membrane remodeling. However, we still lack the high resolution structural data of NDPK in complex with different partners, which is necessary for deciphering the mechanism of these diverse functions. In this review we discuss advances in the structure, folding and stability of NDPKs.
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Johnson, Karyn N., Liang Tang, John E. Johnson, and L. Andrew Ball. "Heterologous RNA Encapsidated in Pariacoto Virus-Like Particles Forms a Dodecahedral Cage Similar to Genomic RNA in Wild-Type Virions." Journal of Virology 78, no. 20 (October 15, 2004): 11371–78. http://dx.doi.org/10.1128/jvi.78.20.11371-11378.2004.
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ABSTRACT The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T=3 particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-Å resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.
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Vogel, Dominik, Sigurdur Rafn Thorkelsson, Emmanuelle R. J. Quemin, Kristina Meier, Tomas Kouba, Nadja Gogrefe, Carola Busch, et al. "Structural and functional characterization of the severe fever with thrombocytopenia syndrome virus L protein." Nucleic Acids Research 48, no. 10 (April 20, 2020): 5749–65. http://dx.doi.org/10.1093/nar/gkaa253.
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Abstract The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein–RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.