MACCHESS PROGRAM FOR SOLUTION SAXS AND ENVELOPE PHASING

适用于萨克斯管和包络定相解决方案的 MACCHESS 程序

• 批准号: 8171496
• 负责人: Holger Sondermann
• 金额: $ 1.21万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171496
• 关键词: Attention; Buffers; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Data Collection; Data Set; Encapsulated; Ensure; Enzymes; Figs - dietary; Funding; Goals; Grant; HIV Infections; Hand; Handedness; Human; Institution; Investigation; Learning; Left; Leukocytes; Measures; Methods; Mica; Modeling; Molecular; Optics; Pattern; Phase; Procedures; Proteins; Research; Research Personnel; Resolution; Resources; Running; Sampling; Series; Shapes; Simulate; Solutions; Source; Staging; Structure; Techniques; Testing; Time; Tube; United States National Institutes of Health; base; beamline; computer program; computerized data processing; data acquisition; detector; molecular shape; mylar; particle; programs; reconstruction; research study; software development

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. As described in earlier sections, the collaborators on this initiative have proposed challenging structural problems that demand solution SAXS and concomitant advances in phasing methods. A good example is Joseph Wedekind's (U. Rochester) investigation of the structural basis for function of the APOBEC3G (A3G) enzyme in human white blood cells, where it acts to stave off HIV infections. The team is making progress on crystallizing A3G, but needs complementary structural information with the ultimate goal of learning how this important protein responds to regulatory factors that target it for degradation. Wedekind has teamed up with Richard Gillilan to obtain the general shape of a molecule in solution utilizing SAXS. If and when crystals become available, the solution shape information will be used to phase the crystallographic structures. The proposed phasing procedure requires three steps: (1) Obtain a solution SAXS pattern and determine the molecular envelope. (2) If crystals exist, collect a standard crystallographic data set and use the envelope information for low-resolution phases. (3) Extend phases to higher resolution to solve for the high resolution structure. The procedures are detailed below. Obtain a SAXS Pattern and Determine the Molecular Envelope We propose to optimize experimental techniques to determine molecular envelopes using SAXS. The current setup for SAXS experiments at the CHESS G1 beamline is as follows (Gillilan, 2002, unpublished; Fig. 31): + G1 wiggler line with multilayer optics + 1.2 Angstrom wavelength with ~2% bandpass + Quantum4 2K (ADSC) detector @ ~780mm from sample cell + 500¿m x 500¿m beam with 0.8mm guard slits + He flight tube with Be window on sample end, 0.5mil mylar on detector end + 1mm path (80¿l) sample cell (courtesy of J.G. Grossmann, Daresbury U.K.) + 25¿m mica windows in sample cell Scattering Data Acquisition and Analysis Samples are encapsulated inside a cell sandwiched by two thin parallel windows. SAXS data of buffer and samples at different concentrations are collected. SAXS data are processed using the software developed by Xinguo Hong and Richard Gillilan [108]. The data reduction includes normalization of the scattered data to the intensity of the transmitted beam and subtraction of the background scattering of the buffer. All scattering curves are then standardized to that of a protein concentration of 1 mg/ml. The low angle data will be extrapolated to infinite dilution and merged with the high angle data measured at high protein concentrations to yield final scattering curves. Once the solution scattering data from a protein sample is obtained, the next step is to recover the three-dimensional envelope from the one-dimensional scattering pattern. Two methods developed by Svergun and colleagues will be used. In the first general ab initio approach [74,109], an angular envelope function of the particle, R = F( ), where (r, ) are spherical coordinates, is described by a series of spherical harmonics. The lowresolution shape is thus defined by a few parameters  the coefficients of this series  that fit the scattering data. This approach was implemented in the computer program SASHA [75]. It was demonstrated that, under certain circumstances, a unique envelope can be extracted from the scattering data (except for the handedness of the shape  this ambiguity holds for all ab initio methods in SAXS). Both `left` and `right` hands should be tested and the ambiguity resolved in the later stages of the structure determination, when the hand of helices can be determined. The use of envelopes defined by spherical harmonics is limited to globular particles with relatively simple shapes and without significant internal cavities. More detailed models can be constructed ab initio using different types of Monte Carlo searches and the utilization of a simulated annealing approach in which the shape of the complex is modeled by a large number of close-packed beads, which are moved around so as to match the observed scattering profile as well as possible [110-112]. The Monte-Carlo-based models contain hundreds or thousands of parameters, and caution is required to avoid over-interpretation. A common approach is to align a set of models resulting from independent shape reconstruction runs to obtain an average model that retains the most persistent, and presumably also most reliable, features, e.g. using the program SUPCOMB [113]. Particle symmetry, if known, provides very useful constraints, which can be imposed in the programs SASHA and DAMMIN, and in the program GASBOR [110-112]. Once the molecular shape is determined from the SAXS pattern and crystallographic data are collected, the molecular replacement method implemented in the FSEARCH program [93] is used to locate the envelope in the crystallographic unit cell, thus providing low resolution phases. FSEARCH (distributed by CCP4) can accept either of the two forms of envelope (spherical harmonics or Monte-Carlo-based models) as an input search model. It has been shown that the absence of strong reflections at low resolution caused by saturation at the detector can degrade FSEARCH solutions greatly [114]. Therefore, particular attention is paid in crystallographic data collection experiments to ensure low resolution data (100-10¿) are near complete (by using a small beam stop) and not saturated (by reducing exposure time).

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 如前几节所述，该计划的合作者提出了具有挑战性的结构性问题，需要解决方案 SAXS 以及随之而来的分阶段方法的进步。一个很好的例子是 Joseph Wedekind（U. Rochester）对人类白细胞中 APOBEC 3G (A3G) 酶功能的结构基础的研究，该酶在该酶中发挥作用以避免 HIV 感染。该团队在 A3G 结晶方面取得了进展，但需要补充结构信息，最终目标是了解这种重要的蛋白质如何响应针对其降解的调节因素。 Wedekind 与 Richard Gillilan 合作，利用 SAXS 获得了溶液中分子的一般形状。如果晶体可用，溶液形状信息将用于确定晶体结构的相位。 所提出的定相程序需要三个步骤：(1) 获得解决方案 SAXS 图案并确定分子包膜。 (2) 如果晶体存在，收集标准晶体学数据集并使用低分辨率相的包络信息。 (3) 将相位扩展到更高分辨率以求解高分辨率结构。详细程序如下。 获得 SAXS 模式并确定分子包膜 我们建议优化实验技术，以使用 SAXS 确定分子包膜。 CHESS G1 光束线上 SAXS 实验的当前设置如下（Gillilan，2002 年，未发表；图 31）： + 具有多层光学器件的 G1 摆动线 + 1.2 埃波长，带通约为 2% + Quantum4 2K (ADSC) 探测器@距样品池约 780mm + 500¿m x 500¿m 光束，带 0.8mm 防护狭缝+ 带 Be 窗的 He 飞行管 样品端为 0.5mil 聚酯薄膜，检测器端为 0.5mil 聚酯薄膜 + 1mm 路径 (80¿L) 样品池（由英国 Daresbury 的 J.G. Grossmann 提供）+ 样品池中的 25 µm 云母窗 散射数据采集和分析 样品被封装在由两个平行薄窗夹着的池内。收集不同浓度的缓冲液和样品的 SAXS 数据。 SAXS 数据使用 Xinguo Hong 和 Richard Gillilan 开发的软件进行处理 [108]。数据减少包括将散射数据归一化为透射光束的强度以及减去缓冲器的背景散射。然后将所有散射曲线标准化为 1 mg/ml 蛋白质浓度的散射曲线。低角度数据将外推至无限稀释，并与在高蛋白质浓度下测量的高角度数据合并，以产生最终的散射曲线。 一旦获得蛋白质样品的溶液散射数据，下一步就是从一维散射图案中恢复三维包络。将使用 Svergun 及其同事开发的两种方法。 在第一种通用的从头算方法中[74,109]，粒子的角包络函数 R = F(·)，其中 (r,·) 是球坐标，由一系列球谐函数描述。因此，低分辨率形状由几个参数定义，即适合散射数据的该系列的系数。这种方法在计算机程序 SASHA [75] 中实现。事实证明，在某些情况下，可以从散射数据中提取唯一的包络（除了形状的旋向性之外，这种模糊性适用于 SAXS 中的所有从头计算方法）。应该测试“左”和“右”手，并在结构确定的后期阶段解决歧义，此时可以确定螺旋的手。 由球谐函数定义的包络线的使用仅限于形状相对简单且没有明显内腔的球状颗粒。可以使用不同类型的蒙特卡罗搜索和利用模拟退火方法从头开始构建更详细的模型，其中复合物的形状由大量密堆积珠子建模，这些珠子四处移动以尽可能匹配观察到的散射轮廓[110-112]。基于蒙特卡罗的模型包含数百或数千个参数，需要谨慎避免过度解释。一种常见的方法是对齐由独立形状重建运行产生的一组模型，以获得保留最持久且可能也是最可靠的特征的平均模型，例如使用程序 SUPCOMB [113]。粒子对称性（如果已知）提供了非常有用的约束，可以在程序 SASHA 和 DAMMIN 以及程序 GASBOR 中施加这些约束 [110-112]。 一旦从 SAXS 图案确定了分子形状并收集了晶体学数据，FSEARCH 程序 [93] 中实施的分子替换方法将用于定位晶体晶胞中的包络线，从而提供低分辨率相。 FSEARCH（由 CCP4 分发）可以接受两种形式的包络（球谐函数或基于蒙特卡罗的模型）中的任何一种作为输入搜索模型。事实证明，由于探测器饱和而导致低分辨率下缺乏强反射，会极大地降低 FSEARCH 解决方案的性能 [114]。因此，在晶体学数据收集实验中要特别注意，以确保低分辨率数据 (100-10¿) 接近完整（通过使用小光阑）并且不饱和（通过减少曝光时间）。