Comparison of helical scan and standard rotation methods in single-crystal X-ray data collection strategies

Polsinelli, Ivan; Savko, Martin; Rouanet-Mehouas, C.; Ciccone, Lidia; Nencetti, Susanna; Orlandini, Elisabetta; Stura, E.A.; Shepard, William

doi:10.1107/s1600577516018488

Cited by 26 publications

(29 citation statements)

References 62 publications

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“…[80][81][82] Strategies to mitigate such reduction include merging of composite, low dose partial datasets 83 or helical data collection approaches. 84 Use of rapid X-ray detectors and cryogenic temperatures -typically 100 K -minimises but does not eliminate the effects of photoreduction or radiation damage to crystals. However, intact fully-oxidised structures may require the use of femtosecond X-ray pulses that can only be provided with sufficient brilliance by an X-ray free electron laser (XFEL).…”

Section: Xfel Structures Of Intact Redox States Of Cunirs By Serial Fmentioning

confidence: 99%

Recent structural insights into the function of copper nitrite reductases

et al. 2017

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Copper nitrite reductases (CuNiR) carry out the first committed step of the denitrification pathway of the global nitrogen cycle, the reduction of nitrite (NO) to nitric oxide (NO). As such, they are of major agronomic and environmental importance. CuNiRs occur primarily in denitrifying soil bacteria which carry out the overall reduction of nitrate to dinitrogen. In this article, we review the insights gained into copper nitrite reductase (CuNiR) function from three dimensional structures. We particularly focus on developments over the last decade, including insights from serial femtosecond crystallography using X-ray free electron lasers (XFELs) and from the recently discovered 3-domain CuNiRs.

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Section: Xfel Structures Of Intact Redox States Of Cunirs By Serial Fmentioning

confidence: 99%

Recent structural insights into the function of copper nitrite reductases

et al. 2017

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“…In addition to serial crystallography, which should be used for the smallest crystals, composite data collection for multiple crystals [73] and helical scans for rod shaped crystals [74] are an option to consider. The data collection should have a clear objective and the parameters adjusted accordingly.…”

Section: Data Collection Parametersmentioning

confidence: 99%

Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

Taberman

2018

Crystals

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Radiation damage still remains a major limitation and challenge in macromolecular X-ray crystallography. Some of the high-intensity radiation used for diffraction data collection experiments is absorbed by the crystals, generating free radicals. These give rise to radiation damage even at cryotemperatures (~100 K), which can lead to incorrect biological conclusions being drawn from the resulting structure, or even prevent structure solution entirely. Investigation of mitigation strategies and the effects caused by radiation damage has been extensive over the past fifteen years. Here, recent understanding of the physical and chemical phenomena of radiation damage is described, along with the global effects inflicted on the collected data and the specific effects observed in the solved structure. Furthermore, this review aims to summarise the progress made in radiation damage studies in macromolecular crystallography from the experimentalist's point of view and to give an introduction to the current literature.

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“…Diffraction data were measured at the NSLS-II FXS beamline from LN 2 cryoprotected crystals measuring approximately 50μ X 50μ X 5μ employing a helical data collection mode 13 , using X-rays of 0.9793 wavelength. A Si (111) double crystal monochromator was used to focus the X-ray beam to a spot size of 1μ X 1μ at the sample.…”

Section: Methodsmentioning

confidence: 99%

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

McClelland

Zhang

Mou

et al. 2020

Preprint

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23 USA 24 † Present address, Regeneron Pharmaceutical, Inc. USA 25 26 Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates 27 heterotrimeric G protein alpha subunits (Gα) 1 . Ric-8A is essential to life in 28 multicellular eukaryotes by virtue of its chaperone activity that is required for Gα 29 biogenesis and membrane localization 2,3 . Ric-8A adopts an armadillo (ARM)/HEAT 30 repeat domain architecture and is structurally unrelated to G Protein-Coupled 31 Receptors (GPCR) 4 . Both GEF and chaperone activities are stimulated by Casein 32 Kinase II phosphorylation 5 . The mechanisms by which Ric-8A catalyzes GDP 33 release and GTP binding to Gα, or exerts chaperone activity are unknown. Here, 92 Table 2). Initial crystallographic phases were determined by molecular replacement 93 and subsequently used to fit the cryo-EM density map. Iterative cycles of model-building 94 and refinement utilized both cryo-EM and X-ray diffraction data to generate the final 95 models (Extended Data Table S1 and S2). In the following discussion, we use 96 prefixes "r" and "g" for residue and secondary structure identifiers of Ric-8A and Gα, 97 respectively. ARM/HEAT repeat helices of Ric-8A are designated according to their 98 position in the repeat: "A", "B" or "C" for ARM repeats or "a" and "b" for HEAT repeats, 99 followed by the sequence number of the repeat (1 through 9). We use the established 100 nomenclature for Gα secondary structure [21][22][23] . Descriptions of the crystal structure of 101 Ric-8A:Gα refer to chains A and B, the better ordered of the two complexes in the 102 asymmetric unit. 103The cryo-EM reconstruction reveals Ric-8A residues 2-487 and the whole of Gα with 104 the exception of the disordered linker (residues 50-76) between the Helical and GTPase 105 domains ( Fig. 1a and 1b and Extended Data Video 1). The crystal structure of the Ric- 1068A:Gα complex also reveals continuous electron density for Ric-8A except for the 107 linkage between 422 and 430. Notably, residues C-terminal to the last HEAT repeat 108 (r430-r491) are disordered in structures of Ric-8A in which Gα is absent 4,18 . In the 109 crystal structure, which lacks Nb1056, the helical domain of Gαi1 and its connections to 110 the GTPase domain, including much of gα1 and all of switch I, are disordered. 111Otherwise the cryo-EM and X-ray models of Ric-8A:Gα are in good agreement, although 112 certain irregularly-structured and loop regions in both Gα and Ric-8A show significant 113 divergence (Extended Data Fig. 6). Small angle X-ray scattering (SAXS) 114 measurements of the complex are consistent with the X-ray and cryo-EM structures 115 (Extended Data Fig. 7).116 Figure 2. Interactions of Ric-8A with Gαi1. Three major Ric-8A:Gα contact surfaces and phosphorylation 117 sites are highlighted and labeled in the green, yellow, pink and blue overlays (upper left) and enlarged in 118 panels a-d. Axes adjacent to each panel label indicate the rotation applied to the overall schematic to 119 generate the panel view. a, Interac...

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Comparison of helical scan and standard rotation methods in single-crystal X-ray data collection strategies

Cited by 26 publications

References 62 publications

Recent structural insights into the function of copper nitrite reductases

Recent structural insights into the function of copper nitrite reductases

Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

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