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
基本信息
- 批准号:7955555
- 负责人:
- 金额:$ 1.34万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2009
- 资助国家:美国
- 起止时间:2009-07-01 至 2010-06-30
- 项目状态:已结题
- 来源:
- 关键词:AttentionBuffersCellsComplexComputer Retrieval of Information on Scientific Projects DatabaseDataData CollectionData SetEncapsulatedEnsureEnzymesFigs - dietaryFundingGoalsGrantHIV InfectionsHandHandednessHumanInstitutionInvestigationLearningLeftLeukocytesMeasuresMethodsMicaModelingMolecularOpticsPatternPhaseProceduresProteinsResearchResearch PersonnelResolutionResourcesRunningSamplingSeriesShapesSimulateSolutionsSourceStagingStructureTechniquesTestingTimeTubeUnited States National Institutes of Healthbasebeamlinecomputer programcomputerized data processingdata acquisitiondetectormolecular shapemylarparticleprogramsreconstructionresearch studysoftware development
项目摘要
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).
这个子项目是许多研究子项目中的一个
由NIH/NCRR资助的中心赠款提供的资源。子项目和
研究者(PI)可能从另一个NIH来源获得了主要资金,
因此可以在其他CRISP条目中表示。所列机构为
研究中心,而研究中心不一定是研究者所在的机构。
如前所述,这一倡议的合作者提出了具有挑战性的结构问题,需要解决SAXS和相控方法的相应进步。一个很好的例子是约瑟夫·韦德金德(美国)。罗切斯特)研究APOBEC功能的结构基础人类白色血细胞中的3G(A3 G)酶,它的作用是避免艾滋病毒感染。该团队正在结晶A3 G方面取得进展,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靶向其降解的调控因子。Wedekind与Richard Gillilan合作,利用SAXS获得了溶液中分子的一般形状。如果晶体变得可用,则溶液形状信息将用于晶体结构的定相。 所提出的定相程序需要三个步骤:(1)获得溶液SAXS图谱并确定分子包膜。(2)如果晶体存在,收集标准晶体学数据集,并使用低分辨率相位的包络信息。(3)将相位扩展到更高分辨率以求解高分辨率结构。程序详述如下。 获得SAXS图谱并确定分子包膜我们建议优化使用SAXS确定分子包膜的实验技术。目前在CHESS G1光束线上进行SAXS实验的装置如下(Gillilan,2002年,未发表;图31):+ G1摇摆器线,多层光学器件+ 1.2埃波长,约2%带通+Quantum 4 2K(ADSC)检测器,距离样品池约780 mm + 500 <$m x 500 <$m m光束,带0.8 mm防护狭缝+ He飞行管,样品端带Be窗口,检测器端带0.5 mil聚酯薄膜+1 mm路径(80 <$l)样品池(由J.G.英国达雷斯伯里格罗斯曼公司) + 25¿样品池中的m云母窗散射数据采集和分析样品被封装在两个薄的平行窗口夹在中间的池中。收集不同浓度的缓冲液和样品的SAXS数据。SAXS数据使用Xinguo Hong和Richard Gillilan开发的软件进行处理[108]。数据简化包括将散射数据归一化为透射光束的强度和减去缓冲区的背景散射。然后将所有散射曲线标准化为1 mg/ml蛋白质浓度的散射曲线。将低角度数据外推至无限稀释,并与在高蛋白浓度下测量的高角度数据合并,以产生最终散射曲线。 一旦从蛋白质样品中获得溶液散射数据,下一步就是从一维散射图案中恢复三维包络。Svergun及其同事开发的两种方法将被使用。 在第一种通用从头算方法[74,109]中,粒子的角包络函数R = F(),其中(r,)是球坐标,由一系列球谐函数描述。因此,低分辨率形状由几个参数定义 这个级数的系数 与散射数据吻合这种方法在计算机程序SASHA中实现[75]。结果表明,在某些情况下,可以从散射数据中提取唯一的包络(除了形状的旋向性 这种模糊性适用于SAXS中的所有从头算方法)。“左”和“右”手都应进行测试,并在结构测定的后期阶段解决不确定性,此时可以确定螺旋的手。 由球谐函数定义的包络线的使用仅限于形状相对简单且没有明显内腔的球形颗粒。可以使用不同类型的蒙特卡罗搜索和利用模拟退火方法从头开始构建更详细的模型,其中复合物的形状由大量紧密堆积的珠粒建模,这些珠粒四处移动以尽可能匹配观察到的散射分布[110-112]。基于蒙特-卡罗的模型包含数百或数千个参数,需要谨慎避免过度解释。一种常见的方法是对齐一组由独立形状重建运行产生的模型,以获得保留最持久,也可能是最可靠的特征的平均模型,例如使用程序SUPCOMB [113]。粒子对称性,如果已知的话,提供了非常有用的约束,可以在程序SASHA和DAMMIN以及程序GASBOR [110-112]中施加。 一旦从SAXS图案确定了分子形状并收集了晶体学数据,则使用Festival程序[93]中实施的分子置换方法来定位晶体学晶胞中的包络,从而提供低分辨率相。FRENT(由CCP 4分发)可以接受两种形式的包络(球面谐波或基于蒙特-卡罗的模型)中的任何一种作为输入搜索模型。已经表明,由于检测器饱和而导致的低分辨率下的强反射的缺失会大大降低FRESH解决方案[114]。因此,在晶体学数据收集实验中要特别注意确保低分辨率数据(100-10 <$)接近完整(通过使用小光束光阑)且不饱和(通过减少曝光时间)。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Holger Sondermann其他文献
Holger Sondermann的其他文献
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{{ truncateString('Holger Sondermann', 18)}}的其他基金
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:
8363519 - 财政年份:2011
- 资助金额:
$ 1.34万 - 项目类别:
REGULATION OF MEMBRANE TRAFFICKING BY BAR/F-BAR DOMAIN-CONTAINING PROTEINS
BAR/F-BAR 含结构域蛋白对膜运输的调节
- 批准号:
8169264 - 财政年份:2010
- 资助金额:
$ 1.34万 - 项目类别:
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 - 财政年份:2010
- 资助金额:
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STRUC & MECHANISTIC ANALYSIS OF SIGNALING MOL INVOLVED IN BIOFILM FORMATION
斯特鲁克
- 批准号:
8171495 - 财政年份:2010
- 资助金额:
$ 1.34万 - 项目类别:
STRUC & MECHANISTIC ANALYSIS OF SIGNALING MOL INVOLVED IN BIOFILM FORMATION
斯特鲁克
- 批准号:
7955554 - 财政年份:2009
- 资助金额:
$ 1.34万 - 项目类别:
CYCLIC DI-GMP SIGNALING IN BACTERIAL PATHOGENESIS
细菌发病机制中的环状 DI-GMP 信号传导
- 批准号:
7955190 - 财政年份:2009
- 资助金额:
$ 1.34万 - 项目类别:
STRUCTURE AND PLASTICITY OF PERIPHERAL MEMBRANE PROTEINS
外周膜蛋白的结构和可塑性
- 批准号:
7955188 - 财政年份:2009
- 资助金额:
$ 1.34万 - 项目类别:
STRUCT CHARAC OF CYCLASES & PHOSPHODIESTERASES FROM PSEUDOMONAS AERUGINOSA
环化酶的结构特征
- 批准号:
7721310 - 财政年份:2008
- 资助金额:
$ 1.34万 - 项目类别:
STRUC & MECHANISTIC ANALYSIS OF SIGNALING MOL INVOLVED IN BIOFILM FORMATION
斯特鲁克
- 批准号:
7721309 - 财政年份:2008
- 资助金额:
$ 1.34万 - 项目类别:
Mechanism and regulation of c-di-GMP signaling in bacterial biofilm formation
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- 批准号:
7296012 - 财政年份:2007
- 资助金额:
$ 1.34万 - 项目类别:
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