BEST strategy/
SAD optimization
Gleb BourenkovEMBL-Hamburg
Kappa Workgroup MeetingSeptember 28-29, 2009MAXLAB
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
X-Ray Dose : The energy deposited (via inelastic scattering processes) per mass of a crystal sample Units – Gray (Gy)
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
No significant dependencies on the details of how the dose is deposited –i.e. on the photon flux/exposure time, photon energy, etc.*
(Significant) variation between different crystals ascribed to variation in absorbancee.g. high salt, heavy atom soaks
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 Meeting29/09/2009
1.5 MGy 7 MGy 30 MGy
One and the same crystal sample (translated between data sets), measured with different total dose/data set
%Rmrg Rpim Rano CCano 3.8 1.5 3.5 77 9.0 3.0 4.7 6116.6 6.0 5.6 36 7.8 3.0 3.9 60
%Rmrg Rpim Rano CCano 6.5 2.9 3.5 4017.9 6.3 5.9 2440.8 18.6 9.6 512.4 4.6 4.5 23
Reso %Rmrg Rpim Rano CCano >4.0 4.8 2.5 3.6 502.6-2.4 21.6 8.3 7.2 202.1-2.0 61.3 23.7 16.4 3All-2.0 7.8 6.7 6.1 30
Lo
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ose
Hig
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ose
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7% 35%70%
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
1. Modeling the data statistics as a function of data collection parameters
2. Modeling the diffraction intensity variation with X-ray dose
Optimum data collection conditions for a particular crystal are assessed via modeling
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
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 Å2 per 1 MGy ● Luzatti isomorphism factor (Log σA) decays by 0.1 Å2 per 1 MGy
β=8π2sAD = 0.95 0.95 1.4 1.3 Å2/MGy
Kmetko et al. (2006)Owen et al. (2006)D ½ = 4.3(±0.3) x107Gy >2.5 Å ↔ β=1.05 Å2
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3.50E+07
4.00E+07
Popov et al. (2006)
Dose
RD
fa
cto
rs,
A2 β= 1.0±0.3 Å2/MGy
α= 0.1±0.03 Å2/MGy
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
The user choice
Space group, Cell parameters, Orientation, Mosaicity
I[(h,k,l), Texposure], Ibackground
I/Sigma
Resolution
SAD data
Dose(Time) Constrains
GeometryDose RateDose Rate
Beamline Flux/BeamCrossection
Reference Frames
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
Signal-to-Noise vs Dose
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0.01 0.1 1 10 100
<I/S
igI>
exposure time [sec/degree]
Instrument limit
Absolute limit
BEST model, damage included
BEST model, no damage
bovine trypsinresolution shell 1.75-1.70 Å180x0.5 degrees oscillation frames at ID29
Dose rate 105 Gy/sec
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
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 Meeting29/09/2009
Native data: requested I/SigI in the last resolution binis a target
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spindle angle (o)
# s
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BEST, strategy+predictionsXDS, data statistics
Total dose 21 MGy
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
SAD optimizationMinimum of RFriedel = <|<E2+/w>-<E2-/w>|> is a target
noise 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 Meeting29/09/2009
SAD optimization: search for an optimal crystal orientationminimal RFriedel vs. Resolution – Orientation - Symmetry
P2
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0 0.05 0.1 0.15 0.2 0.251/d^2
Rfr
iede
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1 1 01 1 1random
4.5 3.2 2.6 2.2 Å
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
Interface:CCP4I BEST
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
Multi-crystal data collection
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
0.0
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Exposure, seconds/degree
<J
>/<
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ma
(J)>
wedge 1
wedge 2
wedge 3
wedge 4
Signal-to-noise target C =2
t1=0.32 s
t2=0.41 s
t3=0.53 s
t4=0.71 s
wedge=5°Δφ=1°resolution shell =1.60Ǻ ÷ 1.55 Ǻ
0.0
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Exposure, seconds/degree
<J>
/<S
igm
a(J)
>
wedge 1
wedge 2
wedge 3
Signal-to-noise target C =2
t1=0.52 s
t2=0.88
wedge=5°Δφ=1°resolution shell =1.55Ǻ ÷ 1.50 Ǻ
Truncate rotation range
Single crystal
Lower the resolution
Multiple crystals
Gleb Bourenkov Kappa Workgroup Meeting29/09/2009
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 Meeting29/09/2009
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)
BACKGROUNDReduced subset, selected orientation
Cross-Screeningnarrow wedge in a selected orientation (low dose)EDNA-improved characterization
quantified non-isomorphism – σA matrix
Joint Strategy