A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases.
A high-throughput robotic system has been developed for crystallizing membrane proteins using lipidic mesophases. It incorporates commercially available components and is relatively inexpensive. The crystallization robot uses standard automated liquid-handlers and a specially built device for accurately and reproducibly delivering nanolitre volumes of highly viscous protein/lipid mesophases. Under standard conditions, the robot uses just 20 nl protein solution, 30 nl lipid and 1 microl precipitant solution. 96 wells can be set up using the robot in 13 min. Trials are performed in specially designed 96-well glass plates. The slim (<2 mm high) plates have exquisite optical properties and are well suited for the detection of microcrystals and for birefringence-free imaging between crossed polarizers. Quantitative evaluation of the crystallization progress is performed using an automated imaging system. The optics, in combination with the slim crystallization plates, enables in-focus imaging of the entire well volume in a single shot such that a 96-well plate can be imaged in just 4.5 min. The performance characteristics of the robotic system and the versatility of the crystallization robot in performing vapor-diffusion, microbatch and bicelle crystallizations of membrane and soluble proteins are described.
Cherezov
Vadim
V
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.
Peddi
Avinash
A
Muthusubramaniam
Lalitha
L
Zheng
Yuan F
YF
Caffrey
Martin
M
D001331:Automation; D003201:Computers; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray / Q000295:instrumentation* / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D012371:Robotics; D012984:Software
D008055:Lipids; D008565:Membrane Proteins