BEST strategy / SAD optimization

1. Kappa Workgroup Meeting September 28-29, 2009 MAXLAB BEST strategy /SAD optimization Gleb Bourenkov EMBL-Hamburg

2. Empirical reference figures for radiation damage : 30 MGy Recommended maximum total dose per data set for data collection Owen et al. PNAS 2006 ~1 MGy kinetic rate of (fast) site-specific damage processes the radiation damage may start affecting anomalous signal Translations to the corresponding exposure times at beamlines are available X-Ray Dose : The energy deposited (via inelastic scattering processes) per mass of a crystal sample Units – Gray (Gy) (Significant) variation between different crystals ascribed to variation in absorbance e.g. high salt, heavy atom soaks No significant dependencies on the details of how the dose is deposited – i.e. on the photon flux/exposure time, photon energy, etc.* Journal of Synchrotron Radiation Special Issues on RD - 2006, 2008 J. Holton's survey on http://biosync.rcsb.org *Storage ring, Monochromatic beam Gleb Bourenkov Kappa Workgroup Meeting

3. One and the same crystal sample (translated between data sets), measured with different total dose/data set 70% 7% 35% 1.5 MGy 30 MGy 7 MGy High dose Low dose Optimized data collection parameters / dose distribution Reso%Rmrg Rpim Rano CCano >4.0 4.8 2.5 3.6 50 2.6-2.4 21.6 8.3 7.2 20 2.1-2.0 61.3 23.7 16.4 3 All-2.0 7.8 6.7 6.1 30 %Rmrg Rpim Rano CCano 3.8 1.5 3.5 77 9.0 3.0 4.7 61 16.6 6.0 5.6 36 7.8 3.0 3.9 60 %Rmrg Rpim Rano CCano 6.5 2.9 3.5 40 17.9 6.3 5.9 24 40.8 18.6 9.6 5 12.4 4.6 4.5 23 Gleb Bourenkov Kappa Workgroup Meeting

4. Optimum data collection conditions for a particular crystal are assessed via modeling 1. Modeling the data statistics as a function of data collection parameters 2. Modeling the diffraction intensity variation with X-ray dose Gleb Bourenkov Kappa Workgroup Meeting

5. Radiation Damage Model (Bulk Xtl MX, Cryo) • Diffraction Intensity is a function of dose I(hkl,Dose)=scale(Dose,|hkl|)*I(hkl)+Δ(Dose) ● overall Debye-Waller factor (B) grows by 1 Å2per 1 MGy ● Luzatti isomorphism factor (Log σA) decays by 0.1 Å2per 1 MGy Owen et al. (2006) D ½ = 4.3(±0.3) x107Gy >2.5 Å ↔ β=1.05 Å2 Popov et al. (2006) Kmetko et al. (2006) β= 1.0±0.3 Å2/MGy RD factors, A2 α= 0.1±0.03 Å2/MGy Dose β=8π2sAD = 0.95 0.95 1.4 1.3 Å2/MGy Gleb Bourenkov Kappa Workgroup Meeting

6. Reference Frames MOSFLM XDS HKL Space group, Cell parameters, Orientation,Mosaicity I[(h,k,l), Texposure], Ibackground The user choice Beamline Flux/BeamCrossection I/Sigma Resolution SAD data Dose(Time) Constrains Geometry RADDOSE Dose Rate Plan(s) of data collection Expected data statistics Gleb Bourenkov Kappa Workgroup Meeting

7. Signal-to-Noise vs Dose bovine trypsinresolution shell 1.75-1.70 Å180x0.5 degrees oscillation frames at ID29Dose rate 105 Gy/sec Gleb Bourenkov Kappa Workgroup Meeting

8. Data Collection with Variable Exposure Time and Oscillation Width • BEST optimizes the data collection parameters for each crystal orientation (i.e. spindle position reached after exposure to a certain dose) individually; For convenience of data collection/processing the data collection "plan" is smoothed out to produce a small number sub-wedges with varying exposure/oscillation width • Even without taking the Radiation damage into account, this is useful (e.g. severely anisotropic diffraction or long cell edge) • For high-dose data collection, BEST suggest to increase the exposure time gradually during the data collection, in order to compensate the loss of the diffraction signal due to the radiation damage (according to the model-based expectations) and keep signal-to-noise at a required level. Gleb Bourenkov Kappa Workgroup Meeting

9. Native data: requested I/SigI in the last resolution binis a target Total dose 21 MGy BEST, strategy+predictions XDS, data statistics # strong reflections spindle angle (o) Gleb Bourenkov Kappa Workgroup Meeting

10. SAD optimizationMinimum of RFriedel = <|<E2+/w>-<E2-/w>|> is a targetnoise only, no anomalous scattering itself:decay, non-isomorphismexact pair-vice dose differences for Bijvoet mates • Minimal RFriedel vs. Resolution -> relate expected anomalous signal --------------------------------------------- Resolution RFriedel(%) I/Sigma Multiplicity --------------------------------------------- 10.12 0.8 74.1 23.7 6.90 0.8 43.6 23.7 5.34 1.1 48.4 23.0 4.51 1.2 47.5 23.5 3.98 1.6 34.5 20.6 3.60 2.5 22.4 13.9 3.31 4.0 14.0 11.9 3.08 6.6 8.3 7.0 2.89 10.5 5.2 6.1 2.73 15.6 3.7 2.5 2.60 23.0 2.4 3.8 --------------------------------------------- Gleb Bourenkov Kappa Workgroup Meeting

11. 4.5 3.2 2.6 2.2 Å SAD optimization: search for an optimal crystal orientation minimal RFriedel vs. Resolution – Orientation - Symmetry Gleb Bourenkov Kappa Workgroup Meeting

12. Interface:CCP4I BEST Gleb Bourenkov Kappa Workgroup Meeting

13. Multi-crystal data collection Gleb Bourenkov Kappa Workgroup Meeting

14. Single crystal Multiple crystals Lower the resolution Truncate rotation range Gleb Bourenkov Kappa Workgroup Meeting

15. General approach (current view): • All sample pre-screened (versus an incremental approach) • All crystals selected for data collection will be used in the same orientation (versus "random" orientation - single-axis gonio case, or optimal orientation for each) • Additional cross-characterization round to define the non-isomorphism covariance matrix between selected samples Gleb Bourenkov Kappa Workgroup Meeting

16. Workflow Standard pre-screening (low dose) EDNA pre-selection CELL cluster ORIENTATION common accessible orientation space, + optimization SCALE,B-FACTOR S/N level comparable? MOSAICITY (can weaker diffractors "help" stronger ones) BACKGROUND Reduced subset, selected orientation Cross-Screening narrow wedge in a selected orientation (low dose) EDNA-improved characterization quantified non-isomorphism – σA matrix Joint Strategy Gleb Bourenkov Kappa Workgroup Meeting