As an alternative to vapor diffusion and other crystallization methods, the Anatrace Microlytic Crystal Former utilizes the method of free-interface diffusion to increase crystallization returns in an easy to use and efficient manner(1). Comprising 96 individual microchannels the Microlytic Crystal Former is a fully SBS-compliant crystallization plate, meaning that it is compatible with the most common crystallization and imaging robots, as well as manual set-up and inspection. By using the Microlytic Crystal Former you can screen a much greater area of crystallization space on one plate with fewer conditions and less protein consumption.
The end result? An increased probability of crystallization success by at least two-fold over vapor diffusion.
Since the first structure from a crystal grown in the Microlytic Crystal Former was deposited by researchers at SSGCID(2), there have been many recent, exciting results of researchers using the Crystal Former to produce crystals of their challenging proteins:
Eric Sundberg’s lab at the University of Maryland utilized the Crystal Former to improve the diffraction of their crystals of the enzyme EndoS(3, 4). Prior efforts to crystallize this protein with vapor diffusion led to crystals that diffracted poorly to 7.5 Å; however, rescreening with the Crystal Former and the SuperCOMBI Crystallization Screen led to the identification of new crystal forms which diffracted to 1.9 Å.
Published in Nature Structure & Molecular Biology, researchers from the group of Dirk Jan Slotboom at the University of Groningen obtained initial crystals of the of the membrane protein, PnuC, using the Crystal Former(5). From the author: “Most importantly, Crystal Formers offer a very easy way to probe different crystallization space, which is not possible to achieve with other crystallization techniques. Thus, we always add CFs to our crystallization pipeline to increase the odds of getting crystal hits for our difficult targets, which are usually membrane proteins."
At Queen’s University in Ontario, the lab of Zongchao Jia reported the growth of diffraction quality crystals of the protein, LidL, using the Crystal Former(6). Extensive initial crystallization trials were carried out in vapor diffusion with commercially available crystallization screens of 5 different LidL constructs. While some protein crystals were obtained in vapor diffusion, they were consistently too small and of insufficient quality for high resolution data collection. Screening in the Crystal Former led to the identification of a crystal which permitted collection of a complete data set to 2.76 Å.
Other examples of proteins whose initial crystallization conditions identified using the Crystal Former include RNA Polymerase from E. coli (7), and the human 80S ribosome(8). In each of these cases, the initial crystallization condition identified in the Crystal Former was optimized using other crystallization methods.
1) Stojanoff, V. et al. (2011) Acta Cryst Sect F 67, 971-975.
2) Dranow, D. M. et al. (2013) PDB:4N0Q.
3) Trastoy, B. et al. (2013) Acta Cryst Sect F 69, 1405-1410.
4) Trastoy, B. et al. (2014) PNAS 111, 6714-6719.
5) Jaehme, M. et al. (2014) Nat Struct Mol Biol. 21, 1013-1015.
6) Lee, M.J. et al. (2014) Cryst Growth Des. 14, 3179-3181.
7) Degen, D. et al. (2014) Elife 3, e02451.
8) Khatter, H. et al. (2014) Nucleic Acids Research 42, e49.