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UA BIOC 585 - Crystal Growth

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1Crystal GrowthMcPherson, Crystallization of Biological Macromolecules, 1999Handout: Overview of Protein Crystallization Methods, Pat Weber, Methods in Enzymology(1997), 276:13-23.Crystals Grow in Supersaturated SolutionsMcPherson, Crystallization of Biological Macromolecules, 19992Crystallization Factors• Very pure, homogenous material generally needed• Modifications (e.g. phosphorylation, ligand binding, oligomers, etc.) must be uniform• pH, ionic strength, temperature, ligand, and precipitant are typically varied• Typical: 10 mg/ml, pH 7, NH4(SO4)2 or PEG precipitant• Kits, robots and national facilities available: http://www.hamptonresearch.com/http://www.hwi.buffalo.edu/ProductsServices/highthroughput/HighThroug.htmGenerating X-rays – (Rotating) AnodeDrenth, Principles of Protein X-ray Crystallography, 19993Generating X-rays – SynchrotronDrenth, Principles of Protein X-ray Crystallography, 1999 (left)Blow, Outline of Crystallography for Biologists, 2002 (right)Action Photos: Ultra-high Resolution DataBioCars BMC – Argonne National Labs4Diffraction – Random OrientationIntegration softwareDiffraction with Symmetry5Measuring Diffraction Data• Acquire 180 – 720 images (90 – 360 degrees)• Integrate ~100,000 reflections, apply corrections.Correcting Diffraction Data• Systematic errors: crystal absorption, crystal decay, beam fluctuations, beam polarization, etc. • Apply scale factors to minimize deviation among symmetry mates and reduce errors; need 3-5 fold redundancy• Rmerge= ΣhΣi|Ihi-<Ihi>| \ ΣhΣiIhi• ⏐Fhkl⏐α √Ihkl; Place on absolute scale6Example: Thioredoxin• Human thioredoxin crystal (C73S) treated with H2O2• Data measured and reduced in-house• CCP4i used for final corrections and initial model• PDB Entry 1ERT; 105 aaHuman Thioredoxin7Summary Statistics: Multiplicity & CompletionCompleteness vs Resolution--------------------------------------------------------------------------------Resolution Calc Num Num Num Num Num Avg %Comp %Comprange unique obs rejs mults single unique mult shell cumul--------------------------------------------------------------------------------22.37 - 2.91 2044 8319 330 1951 28 1979 4.04 96.8 96.82.91 - 2.31 1993 8148 32 1892 9 1901 4.27 95.4 96.12.31 - 2.02 1968 6534 19 1793 46 1839 3.54 93.4 95.22.02 - 1.83 1950 5711 19 1741 54 1795 3.17 92.1 94.51.83 - 1.70 1935 5258 2 1702 51 1753 3.00 90.6 93.71.70 - 1.60 1961 5075 0 1706 46 1752 2.90 89.3 93.01.60 - 1.52 1950 4840 2 1674 33 1707 2.83 87.5 92.21.52 - 1.45 1937 4409 2 1605 68 1673 2.63 86.4 91.51.45 - 1.40 1945 3483 9 1455 93 1548 2.24 79.6 90.21.40 - 1.35 1940 1960 0 787 194 981 2.00 50.6 86.3--------------------------------------------------------------------------------22.37 - 1.35 19623 53737 415 16306 622 16928 3.15 86.3 86.3Summary Statistics: RmergeRmerge vs Resolution--------------------------------------------------------------------------------Resolution Average Num Num Num I/sig I/sig Rducd Rmerge Rmergerange counts obs rejs mults unavg avg ChiSq shell cumul--------------------------------------------------------------------------------22.37 - 2.91 53123 8319 330 1951 32.4 64.2 1.55 0.022 0.0222.91 - 2.31 22461 8148 32 1892 23.3 47.7 1.29 0.029 0.0242.31 - 2.02 15090 6534 19 1793 19.3 36.9 1.16 0.031 0.0252.02 - 1.83 8360 5711 19 1741 14.9 27.2 1.02 0.036 0.0261.83 - 1.70 4441 5258 2 1702 10.9 18.9 0.80 0.044 0.0261.70 - 1.60 3278 5075 0 1706 9.3 15.7 0.79 0.053 0.0271.60 - 1.52 2602 4840 2 1674 8.0 13.4 0.86 0.061 0.0271.52 - 1.45 1664 4409 2 1605 5.7 8.9 1.11 0.093 0.0281.45 - 1.40 1105 3483 9 1455 4.1 5.8 0.87 0.111 0.0281.40 - 1.35 664 1960 0 787 3.0 3.9 0.68 0.140 0.028--------------------------------------------------------------------------------22.37 - 1.35 15334 53737 415 16306 15.9 30.1 1.10 0.028


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