Away from the edge: SAD phasing from the sulfur anomalous signal measured in-house with chromium radiation.

IF 2.2 4区 生物学
Cheng Yang, J. Pflugrath, D. A. Courville, C. Stence, J. Ferrara
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引用次数: 43

Abstract

Anomalous scattering with soft X-ray radiation opens new possibilities in phasing for macromolecular crystallography. Anomalous scattering from S atoms collected on an in-house chromium radiation source (lambda = 2.29 A) was used to phase the X-ray diffraction data of thaumatin (22 kDa) and trypsin (24 kDa) crystals. The contribution to the anomalous term, Deltaf" = 1.14 e(-), from sulfur for Cr Kalpha radiation is doubled compared with that for Cu Kalpha radiation, Deltaf" = 0.56 e(-). The direct-methods programs RANTAN or SHELXD successfully found sulfur positions using data sets with resolution limited to 3.5 A. The statistical phasing program SHARP was used to produce the electron-density maps using the sulfur anomalous signal alone at low resolution ( approximately 3.5 A). An interpretable electron-density map for each structure was obtained solely from the phases derived from single-wavelength anomalous dispersion (SAD) data obtained using Cr Kalpha radiation. Much fewer data (that is, lower redundancy) are required for this sulfur SAD phasing procedure compared with the highly redundant data reported in the sulfur SAD phasing procedure with Cu Kalpha radiation. Cr Kalpha radiation can also improve the strength of anomalous scattering of many other intrinsic elements in macromolecules, such as calcium, zinc and phosphorus, because of the increased Deltaf". Furthermore, the anomalous scattering of selenium is increased substantially from 1.14 e(-) with Cu Kalpha radiation to 2.28 e(-) with Cr Kalpha radiation. In order to measure the small Bijvoet differences accurately, several devices were developed for the experiment, including an Osmic Confocal MaxFlux optic optimized for Cr Kalpha radiation, a helium path and a beam stop. In the cases studied here, radiation damage to the samples and reduction of anomalous signal were observed in some long exposure time data sets. Therefore, an adequate data-collection strategy to maximize the completeness in a short scan range was used in subsequent data collections. The results show that the anomalous signal of S atoms can be collected quickly. Since the absorption of solvent and the loop may no longer be negligible with Cr Kalpha radiation, the orientation of the crystal and exposure time were taken into account in order to minimize the effects of radiation damage and absorption. This experimental study shows that using Cr Kalpha radiation from an in-house rotating-anode X-ray generator can provide sufficient phasing power from sulfur anomalous signals to routinely phase protein diffraction data.
远离边缘:SAD相位从硫磺异常信号测量内部铬辐射。
软x射线辐射的异常散射为大分子晶体学的相位分析开辟了新的可能性。利用内部铬辐射源(λ = 2.29 A)收集的S原子的异常散射,对thumatin (22 kDa)和trypsin (24 kDa)晶体的x射线衍射数据进行了相位分析。硫对Cr辐射异常项δ taf”= 1.14 e(-)的贡献是Cu辐射异常项δ taf”= 0.56 e(-)的两倍。直接方法程序RANTAN或SHELXD使用分辨率限制为3.5 A的数据集成功地找到了硫的位置。使用统计相位程序SHARP在低分辨率(约3.5 A)下仅使用硫异常信号生成电子密度图。仅从使用Cr Kalpha辐射获得的单波长异常色散(SAD)数据得出的相位就可以获得每个结构的可解释电子密度图。与Cu - Kalpha辐射的硫SAD相位过程中报告的高度冗余数据相比,该硫SAD相位过程所需的数据要少得多(即冗余度较低)。Cr Kalpha辐射还可以提高大分子中许多其他固有元素的异常散射强度,如钙、锌和磷,因为增加了δ taf”。此外,硒的异常散射从Cu - Kalpha辐射下的1.14 e(-)增加到Cr - Kalpha辐射下的2.28 e(-)。为了精确测量微小的Bijvoet差异,研究人员为实验开发了几种设备,包括针对Cr Kalpha辐射优化的Osmic共聚焦MaxFlux光学装置、氦路径和光束停止装置。在本研究的案例中,在一些长时间暴露的数据集中观察到辐射对样品的损伤和异常信号的减少。因此,在后续的数据收集中,应采用适当的数据收集策略,在短扫描范围内最大限度地提高数据的完整性。结果表明,该方法可以快速采集到S原子的异常信号。由于溶剂和环的吸收在Cr Kalpha辐射下可能不再是可以忽略不计的,因此考虑了晶体的取向和暴露时间,以尽量减少辐射损伤和吸收的影响。本实验研究表明,利用内部旋转阳极x射线发生器的Cr Kalpha辐射可以从硫异常信号到常规相位蛋白质衍射数据提供足够的相位功率。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
自引率
13.60%
发文量
0
审稿时长
3 months
期刊介绍: Acta Crystallographica Section D welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules or the methods used to determine them. Reports on new structures of biological importance may address the smallest macromolecules to the largest complex molecular machines. These structures may have been determined using any structural biology technique including crystallography, NMR, cryoEM and/or other techniques. The key criterion is that such articles must present significant new insights into biological, chemical or medical sciences. The inclusion of complementary data that support the conclusions drawn from the structural studies (such as binding studies, mass spectrometry, enzyme assays, or analysis of mutants or other modified forms of biological macromolecule) is encouraged. Methods articles may include new approaches to any aspect of biological structure determination or structure analysis but will only be accepted where they focus on new methods that are demonstrated to be of general applicability and importance to structural biology. Articles describing particularly difficult problems in structural biology are also welcomed, if the analysis would provide useful insights to others facing similar problems.
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