The TB structural genomics consortium crystallization facility: towards automation from protein to electron density

The crystallization facility of the TB (Tuberculosis) structural genomics consortium, one of nine NIH sponsored P50 structural genomic centres, provides TB consortium members with automated crystallization, data collection and basic molecular replacement (MR) structure solution up to bias minimized electron density maps. Crystallization setup of up to ten proteins per day follows the CRYSTOOL combinatorial screen protocol using a modular and affordable robotic design with an open architecture. Components include screen preparation, plate setup, automated image acquisition and analysis, and optimisation design. A new 96 well crystallization plate has been designed for optimal robotic handling while maintaining ease of manual crystal harvesting. Robotic crystal mounting, screening, and data collection are conducted in-house and at the Advanced Light Source (ALS) in Berkeley. A simple automated protocol based on MR and homology based structure prediction automatically solves modestly difficult problems. Multiple search models are evaluated in parallel MR and the best multi-segment rigid body refined MR solution is subjected to simulated annealing torsion angle molecular dynamics using CNS, bringing even marginal MR solutions within the convergence radius of the subsequent highly effective bias removal and map reconstruction protocol, Shake&wARP, used to generate electron density for initial rebuilding. Real space correlation plots allow rapid assessment of local structure quality. Modular design of robotics and automated scripts using publicly available programs for structure solution allow for efficient high throughput crystallography - at a reasonable cost.

The high-speed Hydra-Plus-One system for automated high-throughput protein crystallography

An automated high-throughput dispenser has been developed for the setup of protein crystallization trials by vapor diffusion or Microbatch methods. The Hydra^®-Plus-One is composed of a Hydra^®-PP system equipped with a motorized XYZ-platform, 96 precision glass syringes and a single-channel microsolenoid dispenser, which transfers 100nl-50μl of protein solution with an accuracy of > 90% at a speed of 60s per 96 wells. Up to 300μl of premixed cocktails can be aspirated with the 96-syringe-assembly and dispensed into reservoir and droplet wells within 60s. The Hydra^®-Plus-One combines high precision, reliability and speed in a cost-effective high-throughput system ideally suited for protein crystallization