The Cloning, Protein Production, Crystallization, and X-ray Data Collection Core constitute a distributed pipeline for X-ray crystal structure determination. The distributed structure takes advantage of local expertise, with the cloning facility at Texas A&M University, the protein purification facility at Los Alamos National Labs, the crystallization facility at Lawrence Livermore National Laboratory, the nanocrystallization facility at UC Berkeley and the X-ray data collection and analysis core located at the Advanced Light Source. This core group is responsible for the majority of the cloning, purification, crystallization and data collection with the program project. The overall goal of this group of core laboratories is to: 1) provide a rapid access library of expressible clones for structural, biochemical and genetic studies, 2) provide highly purified target proteins with sufficient quantity for structure determination through standardization, automation, and parallelization of protocols 3) conduct crystal screens for crystal studies 4) provide screening solutions for inhibitor screens, 5) collect high-resolution MAD data to solve 30-40 structures per year. The pipeline facilities are capable of conducting large-scale clone preparation using a modified Gateway cloning system (Invitrogen) that incorporates a cleavable fusion tag into the system and allows for expression in both E. coli and M. smegmatis. The protein production facility has the capacity to produce 6-8 highly purified target proteins per week using high yield auto-inducing fermentation, parallel cell lysis, parallel affinity and gel filtration chromatography. The crystallization facility currently has the peak capability to prepare 3000 crystallization experiments daily, which is sufficient to screen 8 proteins per day. Some high value targets cannot be produced in quantities sufficient for standard processing and will be screened for crystallization at the UCB nanocrystallization facility. The UCB nano-crystallization facility has developed the infrastructure to screen 384 conditions each for as many as 100 proteins annually using the Fluidigm screening chips. The screening chip requires <2uL of protein per 96 experiments. The X-ray facility has 2 days per month pre-scheduled beam time at the Advanced Light Source (ALS). This time is sufficient to screen crystals and collect MAD data for the determination of up to 30-40 de novo structures per year. The core labs described in this proposal are essential for the successful completion of the overall goals of this program project.
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