BEST strategy / SAD optimization

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


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

w d

ose

Hig

h d

ose

Opt

imiz

ed d

ata

colle

ctio

n p

aram

ete

rs /

dos

e di

strib

utio

n

0 5 10 15 20 25 30

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0 5 10 15 20 25 30

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5 10 15 20 25 30

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7% 35%70%


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


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

0

2

4

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18

0.00E+00

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

3.00E+07

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


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


Signal-to-Noise vs Dose

0

5

10

15

20

25

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


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.


Native data: requested I/SigI in the last resolution binis a target

0

500

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3500

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4500

40 60 80 100 120 140 160

spindle angle (o)

# s

tro

ng

re

fle

cti

on

s

BEST, strategy+predictionsXDS, data statistics

Total dose 21 MGy


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---------------------------------------------


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 0.25

1/d^2

Rfr

ied

el, %

0 1 01 0 01 1 0Random

P222

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1/d^2

Rfr

ied

el,

%

random1 0 01 1 01 1 1

P4

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1/d^2

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iede

l, %

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P23

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0 0.05 0.1 0.15 0.2 0.251/d^2

Rfr

iede

l, % 1 0 0

1 1 01 1 1random

4.5 3.2 2.6 2.2 Å


Interface:CCP4I BEST


Multi-crystal data collection


0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.01 0.1 1 10 100

Exposure, seconds/degree

<J

>/<

Sig

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

0.5

1.0

1.5

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0.01 0.1 1 10 100

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


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


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

Documents

BEST strategy / SAD optimization