Key slides Holton J. M. and Frankel K. A. (2010) Acta D66, 393408 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) 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 Point Spread Function pixel intensity (ADU) distance from point (mm) re-sampled sum scaled and shifted I ~ g(r 2 +g 2 ) -3/2 g = 30 m Holton J. M. and Frankel K. A. (2010) in preparation Spatial Noise: Q315r vs Pilatus Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation average change in spot intensity (%) distance between spots (mm) Pilatus Q315r anomalous differences typically > 100 mm apart! Radiation damage = Kanzaki force? scaled (sin()/) 2 APE1 Wilson plot resolution () R cryst /R free 0.355/ / /0.407 Tsutakawa et al. (2010) in preparation Simulated diffraction image MLFSOM simulatedreal 20% 2 + 5% 2 = 20.6% 2 R cryst + R merge R cryst The R factor Gap in MX Supporting slides Web calculator for experiment success/failure Holton J. M. and Frankel K. A. (2010) Acta D66, 393408 Where: I DL - average damage-limited intensity (photons/hkl) at a given resolution converting R from m to m, r e from m to , from g/cm 3 to kg/m 3 and MGy to Gy r e - classical electron radius (2.818 x m/electron) h- Plancks constant (6.626 x Js) c- speed of light ( m/s) f decayed - fractional progress toward completely faded spots at end of data set - density of crystal (~1.2 g/cm 3 ) R- radius of the spherical crystal (m) - X-ray wavelength () 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 - Matthewss coefficient (~2.4 3 /Dalton) H- Howellss criterion (10 MGy/) - 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 ( 2 ) - attenuation coefficient of sphere material (m -1 ) en - mass energy-absorption coefficient of sphere material (m -1 ) Theoretical limit: Holton J. M. and Frankel K. A. (2010) Acta D66, 393408 Other radiation damage limits Holton J. M. (2009) J. Synchrotron Rad MW (kDa) Resolution () V M ( 3 /Da) Wilson B ( 2 ) Crystal size (m) No. of xtals n0n0 reference 62 ? [1] [1] ?20* Gonzalez & Nave *35125 Teng & Moffat Glaeser et al x30x Facciotti et al * Sliz et al Coulibaly et. al x1.5x Nelson et al Sawaya et al [2] x5x5 [2]43.6 Li et al Standfuss et al x1x Moukhametzianov et al Schuwirth et al [1] [1] Estimated for 100 unit cell in P with V M = 2.4 [2] [2] Taken from 400 um 3 illuminated volume quoted by Moukhametzianov et al. (2008) and 5 um beam Background level sets needed photons/spot Moukhametzianov et al. (2008). Acta Cryst. D 64, Point-spread function of ADSC detectors realistic PSF no PSF Point Spread Function pixel intensity (ADU) distance from point (mm) re-sampled sum scaled and shifted Gaussians Holton J. M. and Frankel K. A. (2010) in preparation Point Spread Function pixel intensity (ADU) distance from point (mm) re-sampled sum scaled and shifted I ~ r 3 Holton J. M. and Frankel K. A. (2010) in preparation Point Spread Function pixel intensity (ADU) distance from point (mm) re-sampled sum scaled and shifted I ~ g(r 2 +g 2 ) -3/2 g = 30 m Holton J. M. and Frankel K. A. (2010) in preparation 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 pixel intensity (ADU) distance from point (CCD pixels) Holton J. M. and Frankel K. A. (2010) in preparation Optimum exposure time calculator 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) 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 Detector spatial noise dominates anomalous difference errors Optimum exposure time (anomalous differences) Holton J. M. and Frankel K. A. (2010) in preparation Optimum exposure time (anomalous differences) I-I+ 3% 100 photons 10 photons 100 photons Holton J. M. and Frankel K. A. (2010) in preparation Optimum exposure time (anomalous differences) I-I+ 3% 100 photons 14 photons 100 photons Holton J. M. and Frankel K. A. (2010) in preparation Optimum exposure time (anomalous differences) 3% I-I photons 67 photons Holton J. M. and Frankel K. A. (2010) in preparation Optimum exposure time (anomalous differences) 1% I-I+ 20,000 photons 200 photons Holton J. M. and Frankel K. A. (2010) in preparation Minimum required signal (MAD/SAD) Holton J. M. and Frankel K. A. (2010) in preparation Spatial Noise Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise down Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise downup Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise downup R separate Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise oddeven R mixed Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise separate:2.5% Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise separate: mixed: 2.5% 0.9% Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise separate: mixed: 2.5% 0.9% 2.5% % 2 = 2.3% 2 Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise mult > ( ) 2 2.3% Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise mult > ( ) 2 R merge Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation Spatial Noise: Q315r vs Pilatus Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation average change in spot intensity (%) distance between spots (mm) Pilatus Q315r anomalous differences typically > 100 mm apart! Diffraction image simulation for tying it all together Simulated diffraction image MLFSOM simulatedreal Sources of noise photon counting Read-out noise Shutter jitter Beam flicker spot shape radiation damage (N) = sqrt(N) rms 11.5 e-/pixel rms 0.57 ms 0.15 %/Hz pixels? mosaicity? B/Gray? The R-factor Gap MLFSOM Elves R merge = 6% R cryst = 17% R free = 20% multi-conformer PDB file 1H87 The R-factor Gap MLFSOM Elves R merge = 6% R cryst = 7% R free = 8% multi-conformer PDB file 1H87 The R-factor Gap MLFSOM Elves R merge = 6% R cryst = 7% R free = 8% single-conformer PDB file 1H87; conf A Sources of noise photon counting Read-out noise Shutter jitter Beam flicker spot shape radiation damage (N) = sqrt(N) rms 11.5 e-/pixel rms 0.57 ms 0.15 %/Hz pixels? mosaicity? B/Gray? Where is the rest of it? 20% 2 + 5% 2 = 20.6% 2 R cryst + R merge R cryst Radiation damageHowells et al. (2009) J. Electron. Spectrosc. Relat. Phenom resolution () maximum tolerable dose (MGy) Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom resolution dependence of radiation damage resolution () maximum tolerable dose (MGy) Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom resolution dependence of radiation damage 10 MGy/ what the is a MGy?damage_rates.pdf Holton J. M. (2009) J. Synchrotron Rad Radiation Damage Model I- average observed spot intensity I 0 - intensity of undamaged spot dose- absorbed dose (MGy) H - 10 MGy/ d- resolution of spot () I = I 0 exp(-ln(2) ) global (lattice) damage dose dH Radiation Damage Model I- average observed spot intensity I 0 - intensity of undamaged spot dose- absorbed dose (MGy) H - 10 MGy/ d- resolution of spot () I = I 0 exp(-ln(2) ) global (lattice) damage dose dHdH 10 MGy/ Radiation Damage Model accumulated dose (MGy) normalized total intensity Radiation Damage Model accumulated dose (MGy) normalized total intensity accumulated dose (MGy) relative B factor data taken from Kmetko et. al Radiation Damage Model accumulated dose (MGy) relative B factor data taken from Kmetko et. al Radiation Damage Model accumulated dose (MGy) relative B factor data taken from Kmetko et. al Radiation Damage Model I- average observed spot intensity I 0 - intensity of undamaged spot dose- absorbed dose (MGy) H - 10 MGy/ d- resolution of spot () I = I 0 exp(-ln(2) ) global (lattice) damage dose dH Radiation Damage Model F- rms observed structure factor F 0 - F of undamaged crystal dose- absorbed dose (MGy) H - 10 MGy/ s- 0.5/d d- resolution of spot () F = F 0 exp(-ln(2) s ) global (lattice) damage dose H 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 () F = F 0 exp( - Bs 2 ) global (lattice) damage Radiation Damage Model F- rms observed structure factor F 0 - F of undamaged crystal A- ln(2)*dose/H H - 10 MGy/ s - 0.5/d d- resolution of spot () F = F 0 exp( - As ) global (lattice) damage Debye-Waller-Ott factor James R. W. (1962) Optical Principles of the Diffraction of X rays. Ox Bow press. 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 () F = F 0 exp( - As - Bs 2 - Cs 3 - ) global (lattice) damage Radiation Damage Model F- rms observed structure factor F 0 - F of undamaged crystal dose- absorbed dose (MGy) H - 10 MGy/ s- 0.5/d d- resolution of spot () F = F 0 exp(-ln(2) s ) global (lattice) damage dose H Radiation Damage Model normalized total intensity Resolution () Gaussian Exponential Reciprocal Space Radiation Damage Model normalized number of atoms magnitude of displacement () Lorentzian Gaussian Direct Space 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? Macroscopic damage crystal expansion Protein crystal in sucrose, NaWO4 and oil crystal expansion Protein crystal in sucrose, NaWO4 and oil crystal expansion Protein crystal in sucrose, NaWO4 and oil crystal expansion Protein crystal in sucrose, NaWO4 and oil Distention of cryo with dose before Distention of cryo with dose after Leapman, R. D. & Sun, S. (1995). Ultramicroscopy, 59, 7179. Distention of cryo with dose High pressure hydrogen bubbles Radiation Damage Model Kanzaki 1957 Radiation Damage Model Kanzaki 1957 stress and strain intensity resolution () stress and strain intensity resolution () stress and strain R bubble R sphere t skin 4/3 R bubble 3 = 4 R sphere 2 t skin u x = dR R x /R sphere F = 8 Y R sphere dR dt/t skin = 2 dR/R sphere P bubble = 2/3 Y stress and strain normalized number of atoms magnitude of displacement (fractional) stress and strain normalized number of atoms magnitude of displacement (fractional) stress and strain normalized number of atoms magnitude of displacement (fractional) stress and strain scaled (sin()/) 2 APE1 Wilson plot resolution () R cryst /R free 0.355/0.514 scaled (sin()/) 2 APE1 Wilson plot resolution () R cryst /R free 0.355/ / /0.407 scaled (sin()/) 2 APE1 Wilson plot resolution () R cryst /R free 0.355/ / /0.407