1. Beamline 8.3.1 summary 1.Strong PRT and staff 2.Robust optics and endstation 3.Safety: stable, simple operations 4.Funding: Operational funding secure 5.Scientific productivity: high 6.Future: streamlining success

2. Holton J. M. (2009) J. Synchrotron Rad. 16 133-42 ALS beamline 8.3.1 Diffraction Methods Research

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

4. 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

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

6. Radiation Damage Model accumulated dose (MGy) best-fit B factor Kmetko et. al. (2006): lysozyme: 0.012 apoferritin: 0.017 slopes (Å 2 /MGy): lysozyme: 0.013 apoferritin: 0.016

7. Simulated diffraction image MLFSOM simulatedreal

8. Crystal Size crystal size (μm) CC to correct model predicted Glaeser et.al. (2000) 1 μm amyloids Nelson et al. 2005 Sawaya et al. 2007 Glaeser et.al. (2000) Sliz et.al. (2003) ~12 μm xylanase Moukhametzianov et al. 2008 5 μm cypovirus polyhedra Coulibaly et. al. 2007 5 μm (13x) bovine rhodopsin Standfuss et al. 2007 theoretical http://bl831.als.lbl.gov/~jamesh/xtalsize.html

9. Minimum Crystal Size n xtal - number of crystals needed n 0 - empirical constant (~ 3) d- d-spacing of interest (Å) B- Wilson B factor (Å 2 ) n xtal = n 0 MW V M 2 ℓ x ℓ y ℓ z (d 3 -1.53) exp(-0.5 B/d 2 ) MW- molecular weight (kDa) V M - Matthews number (~2.5 Å 3 /Da) ℓ- crystal size (microns) B ≈ 4 d 2 + 12 Holton J. M. (2009) J. Synchrotron Rad. 16 133-42 http://bl831.als.lbl.gov/~jamesh/xtalsize.html

10. 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 D submitted

11. Theoretical limit: at ~2.4 Å photon spot μm 3 1.0 Holton J. M. and Frankel K. A. (2010) Acta D submitted for lysozyme

12. 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

13. Specific Damage

14. Damage changes absorption spectrum Photon energy (eV) counts 1 0 Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

15. fluorescence probe for damage fluence (10 15 photons/mm 2 ) Fraction unconverted 25mM SeMet in 25% glycerol 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 250 300 350 400 Exposing at 12680 eV Se cross-section at 12680 eV Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

16. fluorescence probe for damage Absorbed Dose (MGy) Fraction unconverted Wide range of decay rates seen 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 Half-dose = 41.7 ± 4 MGy “GCN4” in crystal Half-dose = 5.5 ± 0.6 MGy 8 mM SeMet in NaOH Protection factor: 660% ± 94% Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

18. Take-home lesson: radiation damage to metal sites is unpredictable Best strategy: 5 MGy to complete data geometrically increasing exposure Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

19. Minimum required signal (MAD/SAD)

20. dataset12-1112 exposure1.0s0.1s1.0s frames100100 x 10100 R merge 5.6% 11.2% 4.7% R anom 4.8%4.7% I/sd 29.5 43.4 33.3 I/sd (2.0 Ǻ)23.3 29.6 25.8 redundancy7.675.77.6 PADFPH36.6937.1137.93 FOM0.3420.3430.366 FOMDM0.6980.7110.726 CC(1H87) 0.418 0.492 0.468 same total dose with high and low redundancy

21. Spatial Noise downup R separate

22. Spatial Noise oddeven R mixed

23. Spatial Noise separate: mixed: 2.5% 0.9% 2.5% 2 -0.9% 2 = 2.3% 2

24. Spatial Noise mult > ( — ) 2 2.3%

25. Minimum Crystal Size n xtal - number of crystals needed n 0 - 3 for complete data set, 180 for MAD d- d-spacing of interest (Å) B- Wilson B factor (Å 2 ) n xtal = n 0 MW V M 2 ℓ x ℓ y ℓ z (d 3 -1.53) exp(-0.5 B/d 2 ) MW- molecular weight (kDa) V M - Matthews number (~2.5 Å 3 /Da) ℓ- crystal size (microns) B ≈ 4 d 2 + 12 Holton J. M. (2009) J. Synchrotron Rad. 16 133-42 http://bl831.als.lbl.gov/~jamesh/xtalsize.html

26. Take-home lesson: need better crystals for MAD Best strategy: find them

27. accurate, unattended data colleciton

28. beamline microscope reference image Re-centering

30. accurate, unattended screening

31. sample shadow on detector Cu

32. sample shadow on detector

33. X-ray shadow of cryo stream

34. Plate goniometers