Key slides

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

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

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 μm 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(θ)/λ) 2 APE1 Wilson plot resolution (Å) 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, 393–408

Where:  I  DL - average damage-limited intensity (photons/hkl) at a given resolution 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 m/electron) h- Planck’s constant (6.626 x J∙s) c- speed of light ( 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

Other radiation damage limits Holton J. M. (2009) J. Synchrotron Rad MW (kDa) Resolution (Å) V M (Å 3 /Da) Wilson B (Å 2 ) Crystal size (μm) 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 Å 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 μm 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) 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

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 σ(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 σ(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 damage Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom

resolution (Å) maximum tolerable dose (MGy) Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom resolution dependence of radiation damage

resolution (Å) maximum tolerable dose (MGy) Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom resolution dependence of radiation damage

10 MGy/Å 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/Å d- resolution of spot (Å) I = I 0 exp(-ln(2) ) global (lattice) damage dose d∙H

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

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/Å d- resolution of spot (Å) I = I 0 exp(-ln(2) ) global (lattice) damage dose d∙H

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

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

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

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 (Å) F = F 0 exp( - A∙s - B∙s 2 - C∙s 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/Å s- 0.5/d d- resolution of spot (Å) F = F 0 exp(-ln(2) s ) global (lattice) damage dose H

Radiation Damage Model normalized total intensity Resolution (Ǻ)  Gaussian Exponential Reciprocal Space

Radiation Damage Model normalized number of atoms magnitude of displacement (Å) 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, 71–79. Distention of cryo with dose High pressure hydrogen bubbles

Radiation Damage Model

Kanzaki 1957

Radiation Damage Model Kanzaki 1957

stress and strain intensity resolution (Å) 

stress and strain intensity resolution (Å) 

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

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(θ)/λ) 2 APE1 Wilson plot resolution (Å) R cryst /R free 0.355/0.514

scaled (sin(θ)/λ) 2 APE1 Wilson plot resolution (Å) R cryst /R free 0.355/ / /0.407

scaled (sin(θ)/λ) 2 APE1 Wilson plot resolution (Å) R cryst /R free 0.355/ / /0.407