Nano liter scale protein crystallization and development of an automatic crystal appraisal method

本研究的成果之一是研制了纳升级的高速半自动蛋白质结晶设备。该器械使用96格式Hydra分配器开发。手动设置400个1或2 μ L规模的不同结晶实验需要半天以上的时间，但使用该设备，可以在大约4分钟内设置96个500 nL的结晶液滴。现在我们可以用少量的蛋白质样品尝试更多的结晶条件，新的结晶板也已经开发出来。该板允许收集液滴中晶体的振荡照片。通常，晶体的X射线衍射质量与晶体的外观不成比例。通常，应从结晶液滴中取出一个晶体，用X射线照射以检查其衍射能力。然而，从水滴中拾取晶体是一个困难的过程。这也需要时间，有时水晶会受到相当大的物理伤害。专门的结晶微孔板可以通过X射线直接检测液滴中的晶体。板上的液滴可以以任何板取向暴露于X射线。有了这个板，我们可以在没有任何物理扰动的情况下检查晶体的固有衍射质量。此外，该微孔板还可用于悬滴和坐滴气相扩散法。结合改进的Hydra 96分配器，该微孔板可实现蛋白质结晶的高通量筛选。这些设备将被用作我们实验室目前正在进行的蛋白质3000项目的重要组成部分。

One of the results of this research was development of high-speed and semi-automatic protein crystallization equipment of nano-liter scale. The device was developed using 96 format Hydra dispenser. It took more than half a day to setup 400 different crystallization experiments of 1 or 2 μL scale by hand, but with this device it become possible to setup 96 crystallization drops of 500 nL in about four minutes. Now we can try more crystallization conditions with few amount of protein sample.New crystallization plate has also been developed. The plate allows collecting an oscillation photograph of the crystal in the drop as it is. Generally, the X-ray diffraction-quality of the crystal is not proportional to the appearance of a crystal. Usually one crystal should be picked up from the crystallization drop to be exposed to X-ray to check its diffraction power. However, picking up a crystal from the drop is kind of a difficult process. It also takes time, and sometimes a crystal receives considerable physical damage. The specialized crystallization microplate makes it possible to inspect crystals in the drop directly by X-ray. The drop on the plate can be exposed to X-ray with any plate orientation. With this plate, we can check the intrinsic diffraction quality of crystals without any physical perturbations. In addition, the plate can be used for both hanging and sitting drop vapor diffusion method.In combination with the modified Hydra 96 dispenser, this microplate enables high-throughput screening of crystallization of proteins. These devices will be used as important components of the protein 3000 project that is currently carried out at our laboratory.