Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography

Foadi, James; Aller, Pierre; Alguel, Yilmaz; Cameron, Alexander D.; Axford, Danny; Owen, Robin L.; Armour, Wes; Waterman, David; Iwata, So; Evans, Gwyndaf

doi:10.1107/s0907444913012274

Cited by 242 publications

(207 citation statements)

References 29 publications

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“…CF 2 data were collected at beamline I24 of the Diamond Light Source and showed quick radiation damage. To obtain a full data set, the first five degrees of data from nine crystals were combined in the Blend program (27) after processing with the XDS package (28). The structure was solved in the Phaser program (29) with a monomer of M, as solved previously (25), as the search model (PDB accession no.…”

Section: Methodsmentioning

confidence: 99%

Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

Förster

Maertens

Farrell

et al. 2015

J Virol

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Respiratory syncytial virus (RSV) infects epithelial cells of the respiratory tract and is a major cause of bronchiolitis and pneumonia in children and the elderly. The virus assembles and buds through the plasma membrane, forming elongated membrane filaments, but details of how this happens remain obscure. Oligomerization of the matrix protein (M) is a key step in the process of assembly and infectious virus production. In addition, it was suggested to affect the conformation of the fusion protein, the major current target for RSV antivirals, in the mature virus. The structure and assembly of M are thus key parameters in the RSV antiviral development strategy. The structure of RSV M was previously published as a monomer. Other paramyxovirus M proteins have been shown to dimerize, and biochemical data suggest that RSV M also dimerizes. Here, using size exclusion chromatography-multiangle laser light scattering, we show that the protein is dimeric in solution. We also crystallized M in two crystal forms and show that it assembles into equivalent dimers in both lattices. Dimerization interface mutations destabilize the M dimer in vitro. To assess the biological relevance of dimerization, we used confocal imaging to show that dimerization interface mutants of M fail to assemble into viral filaments on the plasma membrane. Additionally, budding and release of virus-like particles are prevented in M mutants that fail to form filaments. Importantly, we show that M is biologically active as a dimer and that the switch from M dimers to higher-order oligomers triggers viral filament assembly and virus production. IMPORTANCEHuman respiratory syncytial virus (RSV) is the most frequent cause of infantile bronchiolitis and pneumonia. The enormous burden of RSV makes it a major unmet target for a vaccine and antiviral drug therapy. Oligomerization of the matrix protein is a key step in the process of assembly and production of infectious virus, but the molecular mechanism of RSV assembly is still poorly understood. Here we show that the RSV matrix protein forms dimers in solution and in crystals; the dimer is essential for formation of higher-order oligomers. Destabilizing the dimer interface resulted in the loss of RSV filament formation and a lack of budding of virus-like particles. Importantly, our findings can potentially lead to new structure-based RSV inhibitors targeting the assembly process.H uman respiratory syncytial virus (RSV), an enveloped, nonsegmented negative-strand RNA paramyxovirus, is the major worldwide cause of bronchiolitis and pneumonia in infants and of morbidity and mortality in the elderly. Despite the large global impact of RSV infection, no specific antivirals or licensed vaccine is yet available (1, 2). Designing new antivirals requires detailed molecular and structural understanding of key steps in RSV replication.RSV encodes 11 proteins, with the 3 glycoproteins, the fusion protein (F), glycoprotein (G), and small hydrophobic protein (SH), being present in the viral envelope. The virion ...

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Section: Methodsmentioning

confidence: 99%

Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

Förster

Maertens

Farrell

et al. 2015

J Virol

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“…In principle, the ÁCC 1/2 method shares this restriction with the BLEND method (Foadi et al, 2013), which uses a large cell parameter deviation as rejection criterion. However, since it uses the experimental intensity data, the ÁCC 1/2 method directly targets the desired property of optimizing the merged intensity data, and is successful in doing so as seen when being validated.…”

Section: Choice Of Target Function and Rejection Criterionmentioning

confidence: 99%

“…Another method that can be employed is hierarchical clustering of datasets based on their cell parameters (Foadi et al, 2013). This method avoids the problem of false positive rejection of weak datasets, but on the other hand similarity of cell parameters is only a necessary but not a sufficient condition, and does not take similarity of diffraction into account.…”

Section: Introductionmentioning

confidence: 99%

Identification of rogue datasets in serial crystallography

Aßmann¹,

Diederichs²

2015

Acta Cryst Sect A

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Advances in beamline optics, detectors and X-ray sources allow new techniques of crystallographic data collection. In serial crystallography, a large number of partial datasets from crystals of small volume are measured. Merging of datasets from different crystals in order to enhance data completeness and accuracy is only valid if the crystals are isomorphous, i.e. sufficiently similar in cell parameters, unit-cell contents and molecular structure. Identification and exclusion of non-isomorphous datasets is therefore indispensable and must be done by means of suitable indicators. To identify rogue datasets, the influence of each dataset on CC 1/2 [Karplus & Diederichs (2012). Science, 336, 1030-1033], the correlation coefficient between pairs of intensities averaged in two randomly assigned subsets of observations, is evaluated. The presented method employs a precise calculation of CC 1/2 that avoids the random assignment, and instead of using an overall CC 1/2 , an average over resolution shells is employed to obtain sensible results. The selection procedure was verified by measuring the correlation of observed (merged) intensities and intensities calculated from a model. It is found that inclusion and merging of non-isomorphous datasets may bias the refined model towards those datasets, and measures to reduce this effect are suggested.

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“…Advanced experimental work-flows will enable segments of data collected in different geometries, or from different positions, to be combined to generate the best complete data possible. Software to rank and scale together many independent partial data collections is being developed at several sites (Foadi et al, 2013) and will improve with increasingly refined statistical data models. PX operation modes will be flexible and more integrated with down-stream crystallization platforms, which should soon be able to transfer pre-determined sample coordinates to the beamline for analysis.…”

Section: Perspectivesmentioning

confidence: 99%

Generation X-ray – A coming of age

Fox¹

2015

Arbor

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ABSTRACT:In 2014, the scientific community celebrated the impact crystallography has had on fundamental and applied science, and the ground-breaking discoveries that have gifted us with vision into the molecular and nano-worlds. UNESCO has declared 2015 as the International Year of Light, so now is perhaps a well timed moment to reflect on the role synchrotrons play in the field. Access to synchrotron radiation has revolutionized our ability to explore the properties, interactions and structure of materials, and broadened our understanding in diverse scientific arenas ranging from our cultural heritage, to nano-technology and new materials, to cellular systems and drug discovery. The range of potential synchrotron applications and fields of interest is enormous and beyond the scope of any single article, so my aim here, is simply to provide a snapshot filtered through the lens of biological crystallography. We will look back at how synchrotrons developed into the state-ofthe-art facilities we see today, how beamlines adapted to the quickening brought on by the introduction of high throughput techniques at the turn of the millennium, and forward to some of the new directions and technologies that are transforming modern light sources and crystallography.KEYWORDS: beamline; light sources; membrane protein; microfluidics; protein crystallography; serial crystallography; structural biology; synchrotron; XFEL. RESUMEN:La comunidad científica celebró en 2014 el impacto que la cristalografía ha tenido sobre la ciencia fundamental y aplicada, así como los de descubrimientos relevantes que nos han proporcionado una visión del mundo a escala molecular y nanométri-ca. La UNESCO ha declarado 2015 como el Año Internacional de la Luz por lo que quizás es un buen momento para reflexionar sobre el papel que han jugado y juegan los sincrotrones en esta ciencia. El acceso a la radiación sincrotrón ha revolucionado nuestra capacidad para explorar las propiedades, interacciones y estructura de materiales, así como ampliado nuestro conocimiento en distintos contextos científicos, desde el patrimonio cultural a la nanotecnología y nuevos materiales, hasta la exploración de sistemas celulares y búsqueda de fármacos. El rango de potenciales aplicaciones de los sincrotrones y sus campos de interés es vasto y queda fuera del alcance de un único artículo por lo que la intención del autor es la de proporcionar una imagen de los sincrotrones obtenida a través del filtro de la cristalografía macromolecular. Echaremos la vista atrás para ver cómo los sincrotrones han ido evolucionado hasta llegar a las actuales instalaciones en donde las líneas de trabajo asociadas a los mismos se están adaptado rápidamente a la introducción de las técnicas de alto rendimiento en el cambio del milenio; pero también miraremos hacia el futuro, hacia las nuevas tendencias y tecnologías que ya están transformando las actuales instalaciones y la misma cristalografía.PALABRAS CLAVE: biología estructural, cristalografía de proteínas, cristalografía en serie, línea, ...

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Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography

Abstract: A systematic approach to the scaling and merging of data from multiple crystals in macromolecular crystallography is introduced and explained.

Cited by 242 publications

References 29 publications

Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

Identification of rogue datasets in serial crystallography

Generation X-ray – A coming of age

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