X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography

Foos, Nicolas; Seuring, Carolin; Schubert, Robin; Burkhardt, Anja; Svensson, Olof; Meents, A.; Chapman, Henry N.; Nanao, Max H.

doi:10.1107/s2059798318001535

Cited by 10 publications

(7 citation statements)

References 44 publications

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“…In the future, we plan to improve the current 2.7 Å resolution crystal for better diffraction, and determine the high resolution structure of frog EPN by direct phasing. During the process, we plan to exploit conventional metal soaking [22] and other well-known techniques to work such as the use of (Ta 6 Br 12 ) 2+ [23] or 5-amino-2,4,6-triiodoisophthalic acid [24], and even sulfur-SAD [25,26] or ultraviolet radiation damage-induced phasing (UV-RIP) [27] methods. Since no structural information on any EPN exists in databases, the structure would give us insight into understanding its function.…”

Section: Resultsmentioning

confidence: 99%

Over-Expression, Secondary Structure Characterization, and Preliminary X-ray Crystallographic Analysis of Xenopus tropicalis Ependymin

2018

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Abstract:The gene encoding frog (Xenopus tropicalis) ependymin without the signaling sequence was gene-synthesized, and the protein successfully over-expressed in~mg quantities adequate for crystallization using insect cell expression. Circular dichroism (CD) analysis of the protein purified with >95% homogeneity indicated that ependymin contains both α-helix and β-strand among the secondary structure elements. The protein was further crystallized using polyethylene glycol 8000 as the precipitating reagent, and X-ray diffraction data were collected to 2.7 Å resolution under cryo-condition at a synchrotron facility. The crystal belongs to a hexagonal space group P6 1 22 (or P6 5 22) having unit cell parameters of a = b = 61.05 Å, c = 234.33 Å. Matthews coefficient analysis indicated a crystal volume per protein mass (V M ) of 2.76 Å 3 Da −1 and 55.4% solvent content in the crystal when the calculated molecular mass of the protein only was used. However, the apparent SDS-PAGE molecular mass of~33 kDa (likely resulting from N-glycosylation) suggested V M of 1.90 Å 3 Da −1 and 35.4% solvent content instead. In both cases, the asymmetric unit of the crystal likely contains only one subunit of the protein.

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Section: Resultsmentioning

confidence: 99%

Over-Expression, Secondary Structure Characterization, and Preliminary X-ray Crystallographic Analysis of Xenopus tropicalis Ependymin

2018

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“…This technique can be loosely viewed as an 'inverted' SIR experiment. We have previously shown that radiation-damageinduced phasing is possible in serial experiments (Foos et al, 2018). This work employed a modified MeshAndCollect workflow which repeatedly collected data from the same crystals in order to obtain high-and low-dose data sets.…”

Section: Discussionmentioning

confidence: 99%

Single-support serial isomorphous replacement phasing

Foos¹,

Rizk²,

Nanao³

2022

Acta Cryst Sect D Struct Biol

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The use of single isomorphous replacement (SIR) has become less widespread due to difficulties in sample preparation and the identification of isomorphous native and derivative data sets. Non-isomorphism becomes even more problematic in serial experiments, because it adds natural inter-crystal non-isomorphism to heavy-atom-soaking-induced non-isomorphism. Here, a method that can successfully address these issues (and indeed can benefit from differences in heavy-atom occupancy) and additionally significantly simplifies the SIR experiment is presented. A single heavy-atom soak into a microcrystalline slurry is performed, followed by automated serial data collection of partial data sets. This produces a set of data collections with a gradient of heavy-atom occupancies, which are reflected in differential merging statistics. These differences can be exploited by an optimized genetic algorithm to segregate the pool of data sets into `native' and `derivative' groups, which can then be used to successfully determine phases experimentally by SIR.

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“…Nevertheless, RIP is a powerful tool because the data can be collected from the same crystal at the same position (de Sanctis et al, 2016). Such an approach can also be used in synchrotron serial crystallography (Foos et al, 2018).…”

Section: Uv-rip: Thaumatinmentioning

confidence: 99%

SHELIXIR: automation of experimental phasing procedures using SHELXC/D/E

et al. 2021

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The program SHELIXIR represents a simple and efficient tool for routine phase-problem solution using data for experimental phasing by the single-wavelength anomalous dispersion, multiwavelength anomalous dispersion, single isomorphous replacement with anomalous scattering and radiation-damage-induced phasing methods. As indicated in its name, all calculation procedures are performed with the SHELXC/D/E program package. SHELIXIR provides screening for alternative space groups, optimal solvent content, and high- and low-resolution limits. The procedures of SHELXE are parallelized to minimize the computational time. The automation and parallelization of such procedures are suitable for phasing at synchrotron beamlines directly or for finding the optimal parameters for further data processing. A simple graphical interface is designed to make use easier and to increase efficiency during beam time.

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X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography

Abstract: Multi-crystal serial crystallography data can be used for UV and X-ray radiation-damage-induced phasing.

Cited by 10 publications

References 44 publications

Over-Expression, Secondary Structure Characterization, and Preliminary X-ray Crystallographic Analysis of Xenopus tropicalis Ependymin

Over-Expression, Secondary Structure Characterization, and Preliminary X-ray Crystallographic Analysis of Xenopus tropicalis Ependymin

Single-support serial isomorphous replacement phasing

SHELIXIR: automation of experimental phasing procedures using SHELXC/D/E

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