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Key slides

Calculate the optimum exposure time for your data set, taking into account background levels, ADC offset, read-out noise, and photon gain. Use this tool to optimize your experiment success.

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Key slides

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  1. Key slides

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

  3. Optimum exposure time(faint spots) thr optimum exposure time for data set (s) tref exposure time of reference image (s) bgref background level near weak spots on reference image (ADU) bg0 ADC offset of detector (ADU) σ0rms read-out noise (ADU) gain ADU/photon m multiplicity of data set (including partials) Short answer: bghr = 90 ADU for ADSC Q315r Holton J. M. and Frankel K. A. (2010) in preparation

  4. 107 106 105 104 103 100 10 1 Point Spread Function re-sampled sum scaled and shifted pixel intensity (ADU) I ~ g(r2+g2)-3/2 g = 30 μm 0.01 0.1 1 2 distance from “point” (mm) Holton J. M. and Frankel K. A. (2010) in preparation

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

  6. Radiation damage = Kanzaki force?

  7. APE1 Wilson plot Rcryst/Rfree 0.355/0.514 0.257/0.449 0.209/0.407 scaled <F2> 4.1 3.5 3.2 2.9 2.7 2.5 2.4 2.2 2.1 resolution (Å) (sin(θ)/λ)2 Tsutakawa et al. (2010) in preparation

  8. Simulated diffraction imageMLFSOM simulated real

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

  10. Supporting slides

  11. Web calculator for experiment success/failure

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

  13. Theoretical limit: Where: IDL - average damage-limited intensity (photons/hkl) at a given resolution 105 - converting R from μm to m, re from m to Å, ρ from g/cm3 to kg/m3 and MGy to Gy re - 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) fdecayed - fractional progress toward completely faded spots at end of data set ρ - density of crystal (~1.2 g/cm3) R - radius of the spherical crystal (μm) λ - X-ray wavelength (Å) fNH - the Nave & Hill (2005) dose capture fraction (1 for large crystals) nASU - number of proteins in the asymmetric unit Mr - molecular weight of the protein (Daltons or g/mol) VM - Matthews’s coefficient (~2.4 Å3/Dalton) H - Howells’s criterion (10 MGy/Å) θ - Bragg angle a2 - number-averaged squared structure factor per protein atom (electron2) Ma - number-averaged atomic weight of a protein atom (~7.1 Daltons) B - average (Wilson) temperature factor (Å2) μ - attenuation coefficient of sphere material (m-1) μen - mass energy-absorption coefficient of sphere material (m-1) Holton J. M. and Frankel K. A. (2010) Acta D66, 393–408

  14. Other radiation damage limits [1] Estimated for 100 Å unit cell in P43212 with VM = 2.4 [2] Taken from 400 um3 illuminated volume quoted by Moukhametzianov et al. (2008) and 5 um beam Holton J. M. (2009) J. Synchrotron Rad.16 133-42

  15. Background level sets needed photons/spot Moukhametzianov et al. (2008). Acta Cryst. D64, 158-166

  16. Point-spread function of ADSC detectors

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

  18. 107 106 105 104 103 100 10 1 Point Spread Function re-sampled sum scaled and shifted pixel intensity (ADU) Gaussians 0.01 0.1 1 2 distance from “point” (mm) Holton J. M. and Frankel K. A. (2010) in preparation

  19. 107 106 105 104 103 100 10 1 Point Spread Function re-sampled sum scaled and shifted pixel intensity (ADU) I ~ r3 0.01 0.1 1 2 distance from “point” (mm) Holton J. M. and Frankel K. A. (2010) in preparation

  20. 107 106 105 104 103 100 10 1 Point Spread Function re-sampled sum scaled and shifted pixel intensity (ADU) I ~ g(r2+g2)-3/2 g = 30 μm 0.01 0.1 1 2 distance from “point” (mm) Holton J. M. and Frankel K. A. (2010) in preparation

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

  22. 105 104 103 pixel intensity (ADU) 100 10 1 distance from “point” (CCD pixels) Holton J. M. and Frankel K. A. (2010) in preparation

  23. Optimum exposure time calculator

  24. Optimum exposure time(faint spots) thr optimum exposure time for data set (s) tref exposure time of reference image (s) bgref background level near weak spots on reference image (ADU) bg0 ADC offset of detector (ADU) σ0rms read-out noise (ADU) gain ADU/photon m multiplicity of data set (including partials) Short answer: bghr = 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. 3% 10 photons 100 photons Optimum exposure time(anomalous differences) I+ I- 100 photons Holton J. M. and Frankel K. A. (2010) in preparation

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

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

  30. 200 photons Optimum exposure time(anomalous differences) 1% I+ I- 20,000 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 down up Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

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

  36. Spatial Noise odd even Rmixed 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% <ΔF/F> Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation

  41. Spatial Noise mult >(—)2 Rmerge <ΔF/F> 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

  45. Spatial Noise

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

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

  48. Diffraction image simulation for tying it all together

  49. Simulated diffraction imageMLFSOM simulated real

  50. “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? Sources of noise

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