Stojanoff Vivian, Jakoncic Jean, Oren Deena A, Nagarajan V, Poulsen Jens-Christian Navarro, Adams-Cioaba Melanie A, Bergfors Terese, Sommer Morten O A
National Synchrotron Light Source, Brookhaven National Laboratories, Upton, NY 11973, USA.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Aug 1;67(Pt 8):971-5. doi: 10.1107/S1744309111024456. Epub 2011 Jul 26.
Advances in automation have facilitated the widespread adoption of high-throughput vapour-diffusion methods for initial crystallization screening. However, for many proteins, screening thousands of crystallization conditions fails to yield crystals of sufficient quality for structural characterization. Here, the rates of crystal identification for thaumatin, catalase and myoglobin using microfluidic Crystal Former devices and sitting-drop vapour-diffusion plates are compared. It is shown that the Crystal Former results in a greater number of identified initial crystallization conditions compared with vapour diffusion. Furthermore, crystals of thaumatin and lysozyme obtained in the Crystal Former were used directly for structure determination both in situ and upon harvesting and cryocooling. On the basis of these results, a crystallization strategy is proposed that uses multiple methods with distinct kinetic trajectories through the protein phase diagram to increase the output of crystallization pipelines.
自动化技术的进步推动了高通量气相扩散法在初始结晶筛选中的广泛应用。然而，对于许多蛋白质而言，筛选数千种结晶条件仍无法获得足够质量的晶体用于结构表征。在此，比较了使用微流控晶体生成器装置和坐滴气相扩散板对奇异果甜蛋白、过氧化氢酶和肌红蛋白进行晶体鉴定的速率。结果表明，与气相扩散相比，晶体生成器能鉴定出更多的初始结晶条件。此外，在晶体生成器中获得的奇异果甜蛋白和溶菌酶晶体可直接用于原位结构测定，以及收获和冷冻冷却后的结构测定。基于这些结果，提出了一种结晶策略，即使用多种方法，通过蛋白质相图中不同的动力学轨迹，以增加结晶流程的产出。
