Layout and Composition of Experimental Station
The high-performance membrane protein crystallography experimental station is equipped with internationally advanced high-precision diffractometer, large-area two-dimensional detector, and automatic sample loading robotic arm (see Figure 2).
Figure 2 Layout and Composition of Experimental Station
Experimental Station Methods and Principles
The membrane protein beamline stands as a pivotal protein crystal diffraction beamline within the Phase II light source project. Its design and construction cater to various protein crystal diffraction experimental methods. In addition to meeting the requirements for conventional-sized protein crystal experiments, it also offers micro-focused beam protein crystallography methods, emphasizing the optimization of microcrystal diffraction experiments and data collection processing capabilities.
1）High-Performance Micro-Focused Beam Protein Crystallography Method
Experimental Principle: Utilizing micron-sized beams matched to the size of protein crystals for X-ray crystallographic structure analysis of small-sized protein crystals and membrane protein crystals.
Performance Indicators:
Photon Energy Range：5-25keV
Focused Beam Size：Continuously adjustable from 0.5-20μm
Sample Point Photon Flux： >1.6×1012phs/s@1×1μm2 ( DMM mode)； >1.6×1011phs/s@1×1μm12 ( DCM mode)
Characteristics and Advantages： Sub-micron focused beam size (~0.5μm), continuously adjustable focused beam size (0.5-20μm), wide photon energy range (5-25keV), and high photon flux（ >1.0×1014phs/s@12keV, DMM mode）.
Application Areas: Structural biology, analysis of microprotein crystals and membrane protein crystal structures.
2）Raster Data Collection and Automatic Data Processing
Principle: Addressing the difficulties in aligning membrane protein microcrystals on the diffractometer and their poor radiation resistance, the sample area is divided into grids equal in size to the beam spot. The raster scanning method is employed to quickly collect diffraction images on each grid. Preliminary processing and analysis of diffraction images are conducted to determine the diffraction capability of each grid. Strong diffraction points within a total rotation angle of 5-10 degrees are selected from several grids to collect 50-100 diffraction data sets. Then, the diffraction data from multiple samples are automatically screened and merged to obtain complete diffraction data.
Performance Indicators: Scanning Speed: 10 grids/s
Data Merging: 200 sets/h
Advantages: The raster method overcomes difficulties in positioning small-sized samples or samples with low contrast under optical microscopes, facilitating diffraction experiments with tiny crystals. Additionally, the raster method, combined with the beamline's robotic arm and diffractometer, can develop fully automated data collection methods, providing more convenience to users. The data merging method automatically analyzes the quality of diffraction data, discarding poor data and retaining good data, enabling users to quickly obtain high-quality diffraction data suitable for structural analysis, thus saving user time and improving sample utilization efficiency.
3）Helical Scan Experiment Method
For crystals larger than the beam size, it's best practice to match the beam size to the crystal size to minimize radiation damage. Helical collection will help mitigate radiation damage by bringing new crystal volumes into the beam for each image. To set up spiral data collection, the start and end points for data collection along the crystal must be defined. This can be accomplished by centering the crystal and saving each point as described above. Then, click and drag the mouse on the region containing two points. This will draw a line between the points defining the data collection axis. Typically, it's better to use two points along the Omega axis, and translation along the perpendicular axis is better than collecting fixed points.
