1. Key slides

2. Holton J. M. and Frankel K. A. (2010) Acta D66, 393–408

3. Optimum exposure time (faint spots) t hr optimum exposure time for data set (s) t ref exposure time of reference image (s) bg ref background level near weak spots on reference image (ADU) bg 0 ADC offset of detector (ADU) σ 0 rms read-out noise (ADU) gain ADU/photon m multiplicity of data set (including partials) Short answer: bg hr = 90 ADU for ADSC Q315r Holton J. M. and Frankel K. A. (2010) in preparation

4. Point Spread Function pixel intensity (ADU) distance from “point” (mm) 10 7 10 6 10 5 10 4 10 3 100 10 1 0.01 0.1 1 2 re-sampled sum scaled and shifted I ~ g(r 2 +g 2 ) -3/2 g = 30 μm Holton J. M. and Frankel K. A. (2010) in preparation

5. Spatial Noise: Q315r vs Pilatus Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation 3.5 3.0 2.5 2.0 1.5 1.0 0.5 average change in spot intensity (%) distance between spots (mm) 0.1 1 10 100 Pilatus Q315r anomalous differences typically > 100 mm apart!

6. Radiation damage = Kanzaki force?

7. scaled (sin(θ)/λ) 2 APE1 Wilson plot 4.1 3.5 3.2 2.9 2.7 2.5 2.4 2.2 2.1 resolution (Å) R cryst /R free 0.355/0.514 0.257/0.449 0.209/0.407 Tsutakawa et al. (2010) in preparation

8. Simulated diffraction image MLFSOM simulatedreal

9. 20% 2 + 5% 2 = 20.6% 2 R cryst + R merge ≈ R cryst The “R factor Gap” in MX

10. Supporting slides

11. Web calculator for experiment success/failure

12. Holton J. M. and Frankel K. A. (2010) Acta D66, 393–408

13. Where:  I  DL - average damage-limited intensity (photons/hkl) at a given resolution 10 5 - converting R from μm to m, r e from m to Å, ρ from g/cm 3 to kg/m 3 and MGy to Gy r e - classical electron radius (2.818 x 10 -15 m/electron) h- Planck’s constant (6.626 x 10 -34 J∙s) c- speed of light (299792458 m/s) f decayed - fractional progress toward completely faded spots at end of data set ρ- density of crystal (~1.2 g/cm 3 ) R- radius of the spherical crystal (μm) λ- X-ray wavelength (Å) f NH - the Nave & Hill (2005) dose capture fraction (1 for large crystals) n ASU - number of proteins in the asymmetric unit M r - molecular weight of the protein (Daltons or g/mol) V M - Matthews’s coefficient (~2.4 Å 3 /Dalton) H- Howells’s criterion (10 MGy/Å) θ- Bragg angle  a 2  - number-averaged squared structure factor per protein atom (electron 2 )  M a  - number-averaged atomic weight of a protein atom (~7.1 Daltons) B- average (Wilson) temperature factor (Å 2 ) μ- attenuation coefficient of sphere material (m -1 ) μ en - mass energy-absorption coefficient of sphere material (m -1 ) Theoretical limit: Holton J. M. and Frankel K. A. (2010) Acta D66, 393–408

14. Other radiation damage limits Holton J. M. (2009) J. Synchrotron Rad. 16 133-42 MW (kDa) Resolution (Å) V M (Å 3 /Da) Wilson B (Å 2 ) Crystal size (μm) No. of xtals n0n0 reference 62 ? [1] [1] 1.92.4?20*3013130 Gonzalez & Nave 1994 141.62.022*35125 Teng & Moffat 2000 282.12.53020112 Glaeser et al. 2000 242.02.5225x30x3059.8 Facciotti et al. 2003 4003.52.565*2019.3 Sliz et al. 2003 28.61.981.5811525.2 Coulibaly et. al. 2007 0.81.31.510 1.5x1.5x5 33.7 Nelson et al. 2005 Sawaya et al. 2007 782.653.065616 [2] x5x5 [2]43.6 Li et al. 2004 733.43.67695133.2 Standfuss et al. 2007 211.52.411.41x1x20903.1 Moukhametzianov et al. 2008 60003.463.470 17180 Schuwirth et al. 2005 [1] [1] Estimated for 100 Å unit cell in P4 3 2 1 2 with V M = 2.4 [2] [2] Taken from 400 um 3 illuminated volume quoted by Moukhametzianov et al. (2008) and 5 um beam

15. Background level sets needed photons/spot Moukhametzianov et al. (2008). Acta Cryst. D 64, 158-166

16. Point-spread function of ADSC detectors

17. “realistic” PSF “no” PSF Point Spread Function

18. pixel intensity (ADU) distance from “point” (mm) 10 7 10 6 10 5 10 4 10 3 100 10 1 0.01 0.1 1 2 re-sampled sum scaled and shifted Gaussians Holton J. M. and Frankel K. A. (2010) in preparation

19. Point Spread Function pixel intensity (ADU) distance from “point” (mm) 10 7 10 6 10 5 10 4 10 3 100 10 1 0.01 0.1 1 2 re-sampled sum scaled and shifted I ~ r 3 Holton J. M. and Frankel K. A. (2010) in preparation

20. Point Spread Function pixel intensity (ADU) distance from “point” (mm) 10 7 10 6 10 5 10 4 10 3 100 10 1 0.01 0.1 1 2 re-sampled sum scaled and shifted I ~ g(r 2 +g 2 ) -3/2 g = 30 μm Holton J. M. and Frankel K. A. (2010) in preparation

21. active area of CCD phosphor sheet severed fibers intact fibers X-ray beam taper-taper barrier spot flood field Holton J. M. and Frankel K. A. (2010) in preparation

22. pixel intensity (ADU) distance from “point” (CCD pixels) 10 5 10 4 10 3 100 10 1 Holton J. M. and Frankel K. A. (2010) in preparation

23. Optimum exposure time calculator

24. Optimum exposure time (faint spots) t hr optimum exposure time for data set (s) t ref exposure time of reference image (s) bg ref background level near weak spots on reference image (ADU) bg 0 ADC offset of detector (ADU) σ 0 rms read-out noise (ADU) gain ADU/photon m multiplicity of data set (including partials) Short answer: bg hr = 90 ADU for ADSC Q315r Holton J. M. and Frankel K. A. (2010) in preparation

25. Detector spatial noise dominates anomalous difference errors

26. Optimum exposure time (anomalous differences) Holton J. M. and Frankel K. A. (2010) in preparation

27. Optimum exposure time (anomalous differences) I-I+ 3% 100 photons 10 photons 100 photons Holton J. M. and Frankel K. A. (2010) in preparation

28. Optimum exposure time (anomalous differences) I-I+ 3% 100 photons 14 photons 100 photons Holton J. M. and Frankel K. A. (2010) in preparation

29. Optimum exposure time (anomalous differences) 3% I-I+ 2000 photons 67 photons Holton J. M. and Frankel K. A. (2010) in preparation

30. Optimum exposure time (anomalous differences) 1% I-I+ 20,000 photons 200 photons Holton J. M. and Frankel K. A. (2010) in preparation

31. Minimum required signal (MAD/SAD) Holton J. M. and Frankel K. A. (2010) in preparation

32. Spatial Noise Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

33. Spatial Noise down Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

34. Spatial Noise downup Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

35. Spatial Noise downup R separate Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

36. Spatial Noise oddeven R mixed Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

37. Spatial Noise separate:2.5% Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

38. Spatial Noise separate: mixed: 2.5% 0.9% Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

39. Spatial Noise separate: mixed: 2.5% 0.9% 2.5% 2 -0.9% 2 = 2.3% 2 Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

40. Spatial Noise mult > ( — ) 2 2.3% Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

41. Spatial Noise mult > ( — ) 2 R merge Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

42. Spatial Noise Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

43. Spatial Noise Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

44. Spatial Noise

46. Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

47. Spatial Noise: Q315r vs Pilatus Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation 3.5 3.0 2.5 2.0 1.5 1.0 0.5 average change in spot intensity (%) distance between spots (mm) 0.1 1 10 100 Pilatus Q315r anomalous differences typically > 100 mm apart!

48. Diffraction image simulation for tying it all together

49. Simulated diffraction image MLFSOM simulatedreal

50. Sources of noise “photon counting” Read-out noise Shutter jitter Beam flicker spot shape radiation damage σ(N) = sqrt(N) rms 11.5 e-/pixel rms 0.57 ms 0.15 %/√Hz pixels? mosaicity? B/Gray?

51. The R-factor Gap MLFSOM Elves R merge = 6% R cryst = 17% R free = 20% multi-conformer PDB file 1H87

52. The R-factor Gap MLFSOM Elves R merge = 6% R cryst = 7% R free = 8% multi-conformer PDB file 1H87

53. The R-factor Gap MLFSOM Elves R merge = 6% R cryst = 7% R free = 8% single-conformer PDB file 1H87; conf “A”

54. Sources of noise “photon counting” Read-out noise Shutter jitter Beam flicker spot shape radiation damage σ(N) = sqrt(N) rms 11.5 e-/pixel rms 0.57 ms 0.15 %/√Hz pixels? mosaicity? B/Gray?

55. Where is the rest of it? 20% 2 + 5% 2 = 20.6% 2 R cryst + R merge ≈ R cryst

56. Radiation damage http://bl831.als.lbl.gov/~jamesh/ribo_blast/ Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom. 170 4-12

57. resolution (Å) maximum tolerable dose (MGy) 1 2 3 5 7 10 20 40 70 100 1 10 100 10 3 Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom. 170 4-12 resolution dependence of radiation damage

58. resolution (Å) maximum tolerable dose (MGy) 1 2 3 5 7 10 20 40 70 100 1 10 100 10 3 Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom. 170 4-12 resolution dependence of radiation damage

59. 10 MGy/Å what the is a MGy? http://bl831.als.lbl.gov/ damage_rates.pdf Holton J. M. (2009) J. Synchrotron Rad. 16 133-42

60. Radiation Damage Model I- average observed spot intensity I 0 - intensity of “undamaged” spot dose- absorbed dose (MGy) H - 10 MGy/Å d- resolution of spot (Å) I = I 0 exp(-ln(2) ) global (lattice) damage dose d∙H

61. Radiation Damage Model I- average observed spot intensity I 0 - intensity of “undamaged” spot dose- absorbed dose (MGy) H - 10 MGy/Å d- resolution of spot (Å) I = I 0 exp(-ln(2) ) global (lattice) damage dose d∙Hd∙H 10 MGy/Å

62. Radiation Damage Model accumulated dose (MGy) normalized total intensity

63. Radiation Damage Model accumulated dose (MGy) normalized total intensity

64. accumulated dose (MGy) relative B factor data taken from Kmetko et. al. 2006 Radiation Damage Model

65. accumulated dose (MGy) relative B factor data taken from Kmetko et. al. 2006 Radiation Damage Model

66. accumulated dose (MGy) relative B factor data taken from Kmetko et. al. 2006 Radiation Damage Model

67. I- average observed spot intensity I 0 - intensity of “undamaged” spot dose- absorbed dose (MGy) H - 10 MGy/Å d- resolution of spot (Å) I = I 0 exp(-ln(2) ) global (lattice) damage dose d∙H

68. Radiation Damage Model F- rms observed structure factor F 0 - F of “undamaged” crystal dose- absorbed dose (MGy) H - 10 MGy/Å s- 0.5/d d- resolution of spot (Å) F = F 0 exp(-ln(2) s ) global (lattice) damage dose H

69. Radiation Damage Model F- rms observed structure factor F 0 - F of “undamaged” crystal B- canonical Debye-Waller factor s - 0.5/d d- resolution of spot (Å) F = F 0 exp( - B∙s 2 ) global (lattice) damage

70. Radiation Damage Model F- rms observed structure factor F 0 - F of “undamaged” crystal A- ln(2)*dose/H H - 10 MGy/Å s - 0.5/d d- resolution of spot (Å) F = F 0 exp( - A∙s ) global (lattice) damage

71. Debye-Waller-Ott factor James R. W. (1962) Optical Principles of the Diffraction of X rays. Ox Bow press.

72. Radiation Damage Model A- something Debye said was zero B- canonical Debye-Waller factor C- something else Debye said was zero s - 0.5/d d- resolution of spot (Å) F = F 0 exp( - A∙s - B∙s 2 - C∙s 3 - … ) global (lattice) damage

73. Radiation Damage Model F- rms observed structure factor F 0 - F of “undamaged” crystal dose- absorbed dose (MGy) H - 10 MGy/Å s- 0.5/d d- resolution of spot (Å) F = F 0 exp(-ln(2) s ) global (lattice) damage dose H

74. Radiation Damage Model normalized total intensity Resolution (Ǻ)  5 2.5 1.7 1.25 1.0 Gaussian Exponential Reciprocal Space

75. Radiation Damage Model normalized number of atoms magnitude of displacement (Å) Lorentzian Gaussian Direct Space

76. Radiation Damage Model How can the distribution of atom displacements from radiation damage NOT be Gaussian? (central limit theorem) what can cause a Lorentzian distribution?

77. Macroscopic damage http://bl831.als.lbl.gov/~jamesh/ribo_blast/

78. crystal expansion Protein crystal in sucrose, NaWO4 and oil

79. crystal expansion Protein crystal in sucrose, NaWO4 and oil

80. crystal expansion Protein crystal in sucrose, NaWO4 and oil

81. crystal expansion Protein crystal in sucrose, NaWO4 and oil

82. Distention of cryo with dose

83. before

84. Distention of cryo with dose after

85. Leapman, R. D. & Sun, S. (1995). Ultramicroscopy, 59, 71–79. Distention of cryo with dose High pressure hydrogen bubbles

86. Radiation Damage Model

90. Kanzaki 1957

91. Radiation Damage Model Kanzaki 1957

92. stress and strain intensity resolution (Å)  10 5 3 2.7 2.5 2.0 1.8 1.5

93. stress and strain intensity resolution (Å)  10 5 3 2.7 2.5 2.0 1.8 1.5

94. stress and strain R bubble R sphere t skin 4/3 π R bubble 3 = 4 π R sphere 2 t skin u x = dR R x /R sphere F = 8π Y R sphere dR dt/t skin = 2 dR/R sphere P bubble = 2/3 Y

95. stress and strain

96. normalized number of atoms magnitude of displacement (fractional) stress and strain

97. normalized number of atoms magnitude of displacement (fractional) stress and strain

98. normalized number of atoms magnitude of displacement (fractional) stress and strain

99. scaled (sin(θ)/λ) 2 APE1 Wilson plot 4.1 3.5 3.2 2.9 2.7 2.5 2.4 2.2 2.1 resolution (Å) R cryst /R free 0.355/0.514

100. scaled (sin(θ)/λ) 2 APE1 Wilson plot 4.1 3.5 3.2 2.9 2.7 2.5 2.4 2.2 2.1 resolution (Å) R cryst /R free 0.355/0.514 0.257/0.449 0.209/0.407

101. scaled (sin(θ)/λ) 2 APE1 Wilson plot 4.1 3.5 3.2 2.9 2.7 2.5 2.4 2.2 2.1 resolution (Å) R cryst /R free 0.355/0.514 0.257/0.449 0.209/0.407