X-ray Screening and Rapid Structure Determination

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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.