X-ray Screening and Rapid Structure Determination

晶体学核心的目的是为所有项目提供机器人结晶和筛选， 使HIV-宿主蛋白复合物结晶，用于HIV-1 Vif、Vpu、Nef、达特和Rev的结构测定 （项目1、3、4、5和6）。第二个功能是在现场或远程访问X射线期间提供帮助 数据收集，或完全“联邦快递”访问Beamline 8.3.1的X射线衍射，和溶液散射访问 Beamline 12.3.1在伯克利的Advanced Light Source（ALS）。可以减少数据帧， 现场确定的结构。第三个功能是提供必要的结构细化， 生物化学家可以在没有任何晶体学过程知识的情况下有效地完成所有步骤。 想要学习或指导晶体学的研究者将可以获得任何必要的培训， 限制.结晶过程由两个两纳升的液滴设置机器人（TTP Mosquitos）促进 - 具有建立LCP实验的能力），其建立结晶试验，通常在基于96孔板的 系统，在4°C和20°C下。将托盘在第二种类型的机器人培养箱中在4°C或20°C下孵育。 系统这些系统能够存储一千个托盘，并在编程的 具有先进光学器件的Formulatrix岩石成像仪，包括交叉偏振和紫外线屏蔽 验证蛋白质晶体的能力。这些图像被储存起来，以便进行远程在线筛选。一旦晶体 可用，核心提供了在伯克利的高级光源（ALS）的晶体筛选， Beamlines 8.3.1.或12.3.1。该系统允许筛选许多水晶环自动化，并返回收集 筛选命中的数据集。Beamline 12.3.1允许完全远程数据收集，也允许 以小角度或广角X射线散射模式（SAXS、WAXS）运行，用于溶液散射。系统在 ALS被保持并发展成为绝大多数蛋白质复合物晶体的前沿技术。 这包括超平面聚焦光学器件和新的Pilatus 3 6 M探测器。可以完成数据处理 同时收集数据。一个有经验的两名成员工作人员可在光束线，使任何HARC 中心成员可以参加使用该系统，返回时带有减少的数据和/或由 异常衍射或分子置换。硒反常衍射 替代或内源硫是可行的。该系统导致了绝大多数艾滋病毒相关的 HARC中心项目团队成员的结构。有经验的调查员可用于 指导或执行细化。该系统可以由没有经验的生物化学家使用， 晶体学，或者可以在每个步骤中根据研究者的优势进行实践。现场培训是 每一步都按需提供。该系统是由詹姆斯霍尔顿和一个 经验丰富的高级工作人员。

The aim of the Crystallography Core is to provide robotic crystallization and screening for all projects that aim to crystallize HIV-host protein complexes for structure determination involving HIV-1 Vif, Vpu, Nef, Tat and Rev (Projects 1, 3, 4, 5 and 6). A second function is to provide assistance during on-site, or remote access X-ray data collection, or entirely ‘Fed-Ex’ access to X-ray diffraction at Beamline 8.3.1, and solution scattering access at Beamline 12.3.1 at the Advanced Light Source (ALS) in Berkeley. Data frames can be reduced and structures determined on-site. A third function is to provide for structure refinement as necessary so that a biochemist can efficiently go through all steps without any knowledge of the crystallographic process. Investigators who want to learn or direct the crystallography will have access to any training necessary, without restriction. The process of crystallization is facilitated by two, two-nanoliter drop setting robots (TTP Mosquitos – one with capacity to set up LCP experiments) that set up crystallization trials, generally in 96 well plate based systems, at both 4°C and at 20°C. The trays are incubated at either 4°C or 20°C in a second type of robotic system. These systems are capable of storing a thousand trays and visualizing them on a programmed schedule (Formulatrix Rock Imager) with advanced optics, including crossed-polarization and UV screening capacity to validate protein crystals. The images are stored for remote screening online. Once crystals are available, the Core provides for screening of crystals at the Advanced Light Source (ALS) in Berkeley at Beamlines 8.3.1. or 12.3.1. The system allows screening many crystal loops robotically, and returning to collect datasets on selected screening hits. Beamline 12.3.1 allows for completely remote data collection, and also for running in a small- or wide-angle X-ray scattering mode (SAXS, WAXS) for solution scattering. The systems at ALS are maintained and evolved to be forefront technology for the vast majority of protein complex crystals. This includes ultra flat focusing optics and a new Pilatus3 6M detector. Data processing can be accomplished in parallel with data collection. An experienced two member staff is available at the beamline so that any HARC Center members may attend to use the system, returning with reduced data and/or a structure determined by anomalous diffraction or molecular replacement, as appropriate. Anomalous diffraction using selenium substitution or endogenous sulfur is feasible. The system has given rise to the vast majority of HIV related structures from members of the HARC Center Project teams. An experienced investigator is available for guidance or execution of refinement. The system can be used by biochemists who have no experience with crystallography, or can be used hands-on at each step as matched to investigator strengths. On-site training is provided at every step as needed. The systems are developed and maintained by James Holton and an experienced senior staff member.