A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results
Brown J, Walter TS, Carter L, Abrescia NG, Aricescu AR, Batuwangala TD, Bird LE, Brown N, Chamberlain PP, Davis SJ, Dubinina E, Endicott J, Fennelly JA, Gilbert RJ, Harkiolaki M, Hon WC, Kimberley F, Love CA, Mancini EJ, Manso-Sancho R, Nichols CE, Robinson RA, Sutton GC, Schueller N, Sleeman MC, Stewart-Jones GB, Vuong M, Welburn J, Zhang Z, Stammers DK, Owens RJ, Jones EY, Harlos K, Stuart DI. (2003), J Appl Cryst. 36, 315-8
An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of X-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full X-ray diffraction data sets have been collected to 3 Å resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.
Key figure: Time-lapse photographs showing the formation of glucose isomerase crystals
Images (a) and (b) show crystals grown in 30% polyethylene glycol 400, 0.1 M Na-HEPES, pH 7.5, 0.2 M magnesium chloride (Hampton research Crystal Screen solution #23), and photographed at t = 0 h and t = 40 h, respectively. Images (c) and (d) show crystals grown in 30% 2-propanol, 0.1 M Tris-HCl, pH 8.5, 0.2 M ammonium acetate (Hampton research Crystal Screen solution #19), and again photographed at t = 0 h and t = 40 h, respectively. Images were taken by the OASIS 1700 image-acquisition system (Veeco, Cambridge, UK).