Robotic nanolitre protein crystallisation at the MRC Laboratory of Molecular Biology.
We have set up high-throughput robotic systems to screen and optimise crystallisation conditions of biological macromolecules with the aim to make difficult structural biology projects easier. The initial screening involves two robots. A Tecan Genesis liquid handler is used to transfer commercially available crystallisation reagents from 15 ml test tubes into the reservoirs of 96-well crystallisation plates. This step is fully automated and includes a carousel for intermediate plate storage, a Beckman plate sealer and a robotic arm, which transfers plates in between steps. For adding the sample, we use a second robot, a 17-tip Cartesian Technologies PixSys 4200 SynQuad liquid handler, which uses a syringe/solenoid valve combination to dispense small quantities of liquid (typically 100 nl) without touching the surface of the plate. Sixteen of the tips are used to transfer the reservoir solution to the crystallisation wells, while the 17th tip is used to dispense the protein. The screening of our standard set of 1440 conditions takes about 3 h and requires 300 microl of protein solution. Once crystallisation conditions have been found, they are optimised using a second Tecan Genesis liquid handler, which is programmed to pipette gradients from four different corner solutions into a wide range of crystallisation plate formats. For 96-well plates, the Cartesian robot can be used to add the sample. The methods described are now used almost exclusively for obtaining diffraction quality crystals in our laboratory with a throughput of several thousand plates per year. Our set-up has been copied in many institutions worldwide.
Stock
Daniela
D
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. stock@mrc-lmb.cam.ac.uk
Perisic
Olga
O
Löwe
Jan
J
D020650:Combinatorial Chemistry Techniques / Q000295:instrumentation* / Q000379:methods; D003460:Crystallization / Q000295:instrumentation* / Q000379:methods; D004739:England; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012371:Robotics / Q000295:instrumentation* / Q000379:methods; D013048:Specimen Handling / Q000295:instrumentation* / Q000379:methods
D046912:Multiprotein Complexes; D011506:Proteins