Research Of Microcrystals Data Collection And Processing Method

Obtaining accurate protein structure information is the basis for protein function studies and structure based small molecule drug design.X-ray crystallography is still the most important method of studying protein structure.X-ray crystallography has an irreplaceable advantage for structure-based small molecule drug design.With the development of synchrotron radiation experimental techniques such as micro-focus beamline stations and fast-readout detectors,it is possible to collect diffraction data using microcrystals.Since small crystals are susceptible to radiation damage,it is difficult to obtain complete structural information from a crystal even at low temperatures.It is necessary to collect multiple sets of diffraction data from dozens to hundreds of crystals and merge multiple data sets to obtain complete structural information.Harvest small crystals are time-consuming and labor-intensive,and it is often need to use X-rays for scanning and positioning microcrystals when collecting data,which will definitely cause radiation damage to small crystals.Therefore,highthroughput sample delivery methods are very important for performing microcrystal data collection experiments at ambient or low temperatures.In this study,we use fixed-target based method to load the crystal sample.We have designed three sample loading methods.The first method is based on quartz chip.Hundreds of crystals are embedded on the grooved quartz chip,which provided a way of high throughput crystal loading and data collection.The crystals are fixed in the groove,which is beneficial to locate the position of the crystals.The quartz chip has a low background scattering,and excess liquid can be removed through the groove structure,which helps to reduce the background scattering caused by the solution.The second method is to grow protein crystals by conventional methods,and then transfer the crystals to a polyimide film for loading.The third method for crystal sample loading is based on in-situ crystal growth and data collection using polyimide films,which do not need to harvest crystals or transfer crystal samples,and the diffraction data are collected with crystals in their growth environment.The soluble protein is directly grown on the polyimide film by a vapor diffusion method and the membrane protein is grown by a lipid cubic phase in polyimide film sandwich.Small film crystallization plates or film sandwiches were sealed in a 24-well plates with crystallization buffer in reservoir to preserve the crystals in humid environment.For the first method,lysozyme microcrystals were used as test samples at microfocus beamline station.By collecting data from multiple crystals,the structure of lysozyme microcrystals with high resolution of 1.39 ? was obtained,which verified the feasibility of this method.Lysozyme and fatty acid-binding protein(FABP4)crystals were used to verify the feasibility of second method.We successfully solved the structure of lysozyme and FABP4 by molecular replacement method,and obtained high quality data which is comparable to the conventional sample mounting method.We used soluble proteins,which are lysozyme and concanavalin A,and a membrane protein of unknown structure as test samples for in-situ crystal growth and data collection experiments to verify the feasibility of the second method.We obtained high resolution structures of lysozyme and concanavalin A at ambient and low temperatures.We screened an unknown structure membrane protein crystallization conditions and obtained crystallization conditions of 6 ? resolution.The method using polyimide films as a crystal plates is suitable for crystallization of soluble proteins and membrane proteins.This in situ data collection method can also be performed at ambient or low temperatures.Due to the high light transmission and low background scattering of the polyimide film,this film-based fixed target method is especially suitable for data collection of small crystals with a size of around 10 microns.