It must be understood that the answers are always relative probabilities based on our current knowledge.
4.6 Data collection aims
• achieve the highest resolution that the crystal will support • measure 100% of all possible reflections • achieve the highest possible data accuracy • collect the data as efficient and fast as possible
4.5 Determine space group
• Systematic absence and reflection conditions: P 21: (0,k,0), k=2n
(0,1,0) (0,3,0) (0,5,0) …
Missing!
Estimation of the number of molecules per unit cell (Z)
• Mount your crystal with a minimum of other material (cryo-protectant, loops, mother-liquor, glass capillaries • Reduce air scatter by placing the crystal close to the collimator, and by placing the beam stop close to the crystal • Make the X-ray beam equal to or smaller than the crystal • Increase the detector distance • Reduce the oscillation width per image
•
4.2 Detector
• Single-Photon Counters:
• low noise; Best for week signals, not for synchrotrons • small active surface • poor spatial resolution
•
• Photographic Film/Image Plates:
diffraction.mpeg
Simulation: XRayView
4.4 Determine unit cell dimensions
• Zero-level plane
RF/CF=PO/CO=PO/(1/ λ)=PO*λ Therefore, PO=RF/(CF* λ) d100=1/PO=(CF* λ)/RF
•
• 优良的脉冲时间结构：其脉冲宽度仅为几十皮秒，可以单束团或多束团模式 运行，相邻脉冲间隔可调为几纳秒至微秒量级，能为研究化学反应动力过程、 生命过程、材料结构变化过程和大气环境污染过程等提供正确可信的数据；
• 高偏振：上海光源中在电子轨道平面上放出的同步光是完全线极化的, 而离 开电子轨道平面方向发射的同步光则是椭圆极化的，因而是研究具有旋光性 的生物分子、药物分子和表现为双色性的磁性材料的有力工具； 准相干：上海光源从插入件引出的高耀度光具有部分相干性, 为众多前沿学 科的显微全息成像分析开辟了道路；
Data collection notes
• Redundancy: High redundancy produces more accurate data and may simplify collection strategy.
Data collection notes
• Exposure time: needs to be long enough to give reasonable statistics at the highest resolution, but not so long as to overload the detector with the strong low angle spots.
Movie examples
• The Importance of Resolution resolution.mpeg • The Importance of Data Completeness completeness.mpeg
4.7 Data collection notes
• Reduce background scattering
•
上海光源的技术特征
• •
全波段：波长范围宽，从远红外直到硬X射线，且连续可调。 高强度：总功率为600千瓦，是Ｘ光机的上万倍。光通量大于1015光子/ （S.10－3bw）。高强度和高通量为缩短实验数据获取时间、进行条件难以控 制的实验以及医学、工业应用提供了可能; 高耀度：其耀度是最强的Ｘ光机的上亿倍，主要光谱复盖区的光耀度为 1017～1020光子/（S.mm2.mrad2.10－3bw）。高亮度为取得突破性科技成果 提供了高空间分辨、高动量分辨和超快时间分辨的条件；
Protein Crystallography
Chapter 4. Data Collection
4.1 Crystal harvesting
• Fishing crystals
Cryo-protectants
• • • • • • Glycerol Ethylene Glycol Sucrose Dimethyl Sulfoxide PEG DDM
Finally, What we get is
作业：
• 思考题：已知unit cell, a=b=60埃，c=108 埃；α=β=90度，γ=120度。Space group: P 63 衍射最高分辩率为1.6埃。蛋白分子量 为26000。问：该晶体的unit cell 中可能含 有多少个蛋白分子。
4.9 Data quality control
or
This global value is not a proper statistical quantifier and is calculated in different ways (Rsym) in different programs.
Higher multiplicity always leads to improved data quality, although it increases the Rmerge factor.
• 简答题：请指出晶体数据收集时，要注意 哪些关键问题。
Movie example
• Cryo-crystallographic technique video
cryocryst_shortvideo.wmv
Programs to be needed
• CCP4i
• Pymol
• Imosflm
• Coot
4.1 X-ray sources
• Sealed X-ray Tubes • Rotating Anode Tubes • Synchrotron Radiation in 1971
or In principle, data contain some information if I/ sigma is higher than 1.0; In the standard applications, the accepted resolution limit is where the I/ sigma falls below about 2.0.
4.3 Diffractometer
• Goniometer head • Goniostat
Movie examples
• This movie is a compilation of actual x-ray diffraction data from a crystal of GCN4-N16A peptide in P3121.
Watch out for radiation damage!
Data collection notes
Mosaicity
Data collection notes Oscillation range
Friedel’sd region
When the crystal has symmetry axes: tilt the symmetry axis by at least theta max from the spindle direction Monoclinic crystals: skew away from the ac plane as well as from the b axis Triclinic crystals: rotation(1st), rotation (2rd, reoriented 2theta max)
• • • • large active surface sufficient spatial resolution relatively affordable readout is too slow at synchrotrons
• Area Detectors :
• • • • Need intense signals sufficient spatial resolution expensive readout is very fast, works at synchrotrons
• Matthews coefficient (Vm):
= cell volume / (molecular weight*Z)
Between : 1.7 and 3.5 (A^3/Dalton)