晶体学课件4

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.
Protein Crystallography
Chapter 4. Data Collection
4.1 Crystal harvesting
• Fishing crystals
Cryo-protectants
• • • • • • Glycerol Ethylene Glycol Sucrose Dimethyl Sulfoxide PEG DDM
• Matthews coefficient (Vm):
= cell volume / (molecular weight*Z)
Between : 1.7 and 3.5 (A^3/Dalton)
Z=N*A A: the number of asymmetric unit in the unit cell
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.
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• 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；
• 简答题：请指出晶体数据收集时，要注意 哪些关键问题。
Movie examples
• The Importance of Resolution resolution.mpeg • The Importance of Data Completeness completeness.mpeg
4.7 Data collection notes
• Reduce background scattering
References:
4.8 Shoot first, ask questions afterward — American procedure
1) Center a crystal randomly oriented in the beam 2) Collect reflections over a few rotations 3) Determine the crystal orientation; index the reflections; determine the unit cell parameters and space group; 4) Decide collection strategy and shoot again 5) Indexing, Scaling and post-refinement of intensity data
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上海光源的技术特征
Hale Waihona Puke Baidu
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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；
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’s law
Data collection notes
Blind 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)
Data collection notes
• Resolution: move detector closer to the detector for high resolution, provided the spots can be resolved.
Beamstop should not block the low resolution spots too.
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.
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
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
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4.2 Detector
• Single-Photon Counters:
• low noise; Best for week signals, not for synchrotrons • small active surface • poor spatial resolution
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• Photographic Film/Image Plates:
• 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
4.5 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)
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。
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
• • • • 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