First Aid

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First Aid PathologyData quality assessment in
PHENIX

• Peter Zwart

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Introduction

• Structure solution can be enhanced by the
  knowledge of the quality and idiosyncrasies of
  the merged data
• Anomalous signal?
• Twinning
• Pseudo centering
• Data characterization should extend beyond
  standard quantities as Rmerge and nominal
  resolution
• A full characterization of a data set might
  provide expert systems, such as wizards, useful
  information on how to most optimally solve a
  structure

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Introduction

• Xtriage is a program that aims to characterize a
  merged X-ray dataset
• Probabilistic unit cell content analyses
• Likelihood based Wilson scaling
• Analyses of mean intensity
• Ice ring detection
• Outlier analyses
• Twinning / pseudo centering
• Anomalous signal

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Likelihood based Wilson Scaling

• Both Wilson B and nominal resolution determine
  the looks of the map
• Zwart Lamzin (2003). Acta Cryst. D50,
  2104-2113.

Bwil 50 Å2 dmin 2Å
Bwil 9 Å2 dmin 2Å
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Likelihood based Wilson Scaling

• Data can be anisotropic
• Traditional straight-line fitting not reliable
  at low resolution
• Solution Likelihood based Wilson scaling
• Results in estimate of anisotropic overall B
  value.
• Zwart, Grosse-Kunstleve Adams, CCP4 newletter,
  2005.

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Likelihood based Wilson Scaling

• Likelihood based scaling not extremely sensitive
  to resolution cut-off, whereas classic straight
  line fitting is.

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Likelihood based Wilson Scaling

• Anisotropy is easily detected and can be
  corrected for.
• Useful for molecular replacement and possibly for
  substructure solution
• Anisotropy correction cleans up your N(Z) plots

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Likelihood based Wilson Scaling

• For the ML Wilson scaling an expected Wilson
  plot is needed
• Obtained from over 2000 high quality experimental
  datasets
• Expected intensity and its standard deviation
  can be obtained

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Likelihood based Wilson Scaling

• Resolution dependent problems can be
  easily/automatically spotted
• Ice rings
• Empirical Wilson plots available for protein and
  DNA/RNA.

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Outlier analyses

• Assume amplitudes are distributed according to
  Wilson distribution
• For a dataset of a given size, the cumulative
  distribution function of the largest E values
  in the dataset can be used to detect outliers

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Pseudo Translational Symmetry

• Can cause problems in refinement and MR
• Incorrect likelihood function due to effects of
  extra translational symmetry on intensity
• Can be helpful during MR
• Effective ASU is smaller is T-NCS info is used.
• The presence of pseudo centering can be detected
  from an analyses of the Patterson map.
• A Fobs Patterson with truncated resolution should
  reveal a significant off-origin peak.

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Pseudo Translational Symmetry

• A database analyses reveal that the height of the
  largest off-origin peaks in truncated X-ray data
  set are distributed according to

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Pseudo Translational Symmetry

• 1-F(Qmax) The probability that the largest off
  origin peak in your Patterson map is not due to
  translational NCS This is a so-called p value
• If a significance level of 0.01 is set, all off
  origin Patterson vectors larger than 20 of the
  height of the origin are suspected T-NCS vectors.

PDBID Height () P-value ()
1sct 77 910-6
1ihr 45 110-3
1c8u 20 1
1ee2 10 5
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Twinning

• Merohedral twinning can occur when the lattice
  has a higher symmetry than the intensities.
• When twinning does occur, the recorded
  intensities are the sum of two independent
  intensities.
• Normal Wilson statistics break down
• Detect twinning using intensity statistics

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Twinning

• Cumulative intensity distribution can be used to
  identify twinning
• (acentric data)
• Pseudo centering
• Normal
• Perfect twin

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Twinning

• Pseudo centering twinning N(Z) looks normal
• Anisotropy in diffraction data produces similar
  trend to Pseudo centering
• Anisotropy can however be removed
• How to detect twinning in presence of T-NCS?
• Partition miller indices on basis of detected
  T-NCS vectors
• Intensities of subgroups follow normal Wilson
  statistics (approximately)
• Use L-test for twin detection
• Not very sensitive to T-NCS if partitioning of
  miller indices is done properly.
• No need to know twin laws not sensitive to
  pseudo symmetry or certain data processing
  problems.

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Twinning
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Twinning

• A data base analyses on high quality, untwinned
  datasets reveals that the values of the first and
  second moment of L follow a narrow distribution
• This distribution can be used to determine a
  multivariate Z-score
• Large values indicate twinning

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Twinning

• Determination of twin laws
• From first principles
• No twin law will be overlooked
• PDB analyses 36 of structures has at least 1
  possible twin law
• 50.9 merohedral 48.2 pseudo merohedral0.9
  both
• 27 of cases with twin laws has intensity
  statistics that warrant further investigation on
  whether or not the data is twinned
• 10 of whole PDB(!)
• Determination of twin fraction
• Fully automated Britton and H analyses as well as
  ML estimate of twin fraction of basis of L
  statistic.

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Conflicting information

• PDBID 1???
• Unit cell 99.5 60.9 70.96 90 134.5 90
• Space group C 2
• Twin laws and estimated twin fractions
• H,-K,-H-L 0.44
• H2L,-K,-L 0.01
• -H-2L, K, HL 0.01
• ltI2gt/ltIgt2 2.10 (theory for untwinned data
  2.0)
• Data does not appear to be twinned
• ltLgt 0.49 (theory for untwinned data 0.5)
• Multivariate Z-score of L test 0.963
• Data does not appear to be twinned

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Conflicting information

• What is going on?
• Estimated twin fraction is large, but data does
  not seem to be twinned
• Twin law H,-K,-H-L is parallel to an existing NCS
  axis
• or
• Twin law H,-K,-H-L is a symmetry axis, and the
  space group is too low
• It should be C2 H,-K,-H-L F222
• http//www.phenix-online.org/cctbx
• Need images to make decision

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Conflicting information

• A DNA example
• Space group P65
• 1 twin law
• Resolution 1.87A
• Native Patterson analyses indicates several
  significant off-origin peaks
• Intensity statistics indicate pseudo translation
  symmetry
• ltI2gt/ltIgt2 4.243
• N(Z) plot not very informative

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Conflicting information

• However
• L test ltLgt0.46
• Data might be twinned.
• Partitioned data might not follow Wilson
  statistics however.
• Britton and H analyses estimate of twin fraction
  is about 40
• Wrong spacegroup?
• Monomer would not fit in ASU
• Twinning, pseudo symmetry, or both?
• Not clear from experimental data only, use
  deposited coordinates
• Rwork28 Rfree34
• Twin fractions via Britton plot
• From Fcalc 11 (due to pseudo symmetry only)
• From Fobs 41 (pseudo symmetry twinning)
• See Lebedev, Vagin, Murshudov (2006) Acta D62,
  83-95.
• Data likely to be twinned.
• Difficult to spot due to TPS and RPS effects on
  intensity statistics

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Anomalous data

• Structure solution via experimental methods
  (especially SAD) is on the rise.
• Presence of anomalous signal indicated by a
  quantity called Measurability
• Fraction of Bijvoet differences for which
• DI/sDIgt3 and (I/sI() and I(-)/sI(-) gt 3)
• Easy to interpret
• At 3 Angstrom 6 of Bijvoet pairs are
  significantly larger than zero

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Anomalous data

• Measurability and ltDI/sDIgt are closely related
• Measurability more directly translates to the
  number of useful Bijvoet differences in
  substructure solution/phasing

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Anomalous data
6 (partially occupied) Iodines in thaumatin at
l1.5Å.
Raw SAD phases, straight after PHASER
A
A B
B
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Anomalous data
6 (partially occupied) Iodines in thaumatin at
l1.5Å.
Density modified phases
A
A B
B
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Anomalous data

• SAD phasing with PHASER
• Very sensitive residual maps
• Residual map indicates where a certain type of
  anomalous scatterers need to be placed to improve
  fit between observed and expected F() and F(-)
• Lysozyme soaked with solution containing
  (NH4)2(OsCl6)
• Wilson B 13.7 dmin1.7
• Data collected at Os L-III edge (fgt10)
• Measurability at 3.0 is 67
• Anomalous signal is strong
• Partial structure is large
• Zheavy2/(Zheavy2Zprotein2)35

PHASER residual map indicating location of main
chain atoms
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Anomalous data

• SAD phasing with PHASER
• Very sensitive residual maps
• Residual map indicates where a certain type of
  anomalous scatterers need to be placed to improve
  fit between observed and expected F() and F(-)
• Lysozyme soaked with solution containing
  (NH4)2(OsCl6)
• Wilson B 13.7 dmin1.7
• Data collected at Os L-III edge (fgt10)
• Measurability at 3.0 is 67
• Anomalous signal is strong
• Partial structure is large
• Zheavy2/(Zheavy2Zprotein2)35

Raw PHASER SAD phases
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Anomalous data

• Another extreme
• 2 Fe4S4 clusters in 60 residues
• Wilson B 6.5Å2 dmin1.2Å
• Measurability at 3.0Å 6
• Data not terribly strong
• ZFe2/(ZFe2ZS2Zprotein2)17
• Fe f 1.25 e S f 0.35 e
• PHASER residual map from Fe SAD phases clearly
  show S positions

SAD on Fe, residual maps indicate S positions
(green balls)
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Anomalous data

• Inclusion of Sulfurs improves phasing
• (ZFe2ZS2)/(ZFe2ZS2Zprotein2)32
• ltFOMgt0.67 (was 0.53)
• Residual maps show almost all non-hydrogen atoms
• Inclusion of non hydrogen atoms results in
  ltFOMgt0.98.

SAD on Fe, S. Residual maps (purple) and FOM
weighted Fobs map (blue).
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Discussion Conclusions

• Software tools are available to point out
  specific problems
• mmtbx.xtriage ltinput_reflection_filegt params
• Log file are not just numbers, but also contains
  an extensive interpretation of the statistics
• Knowing the idiosyncrasies of your X-ray data
  might avoid falling in certain pitfalls.
• Undetected twinning for instance

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First Aid

• Analyses at the beamline
• If problem are detected while at the beam line,
  possible problems could be solved by recollecting
  data or adapting the data collection strategy.

The Surgeon and the Peasant 1524. Lucas van
Leyden
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Pathology/Autopsy

• Analyses at home

The anatomical lesson of dr. Nicolaes Tulp -
1632. Rembrandt van Rijn.
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Ackowledgements
Cambridge Randy Read Airlie McCoy Laurent
Storoni Los Alamos Tom Terwilliger Li Wei
Hung Thirumugan Rhadakanan Texas AM
Univeristy Jim Sacchettini Tom Ioerger Eric
McKee

• Paul Adams
• Ralf Grosse-Kunstleve
• Pavel Afonine
• Nigel Moriarty
• Nick Sauter
• Michael Hohn

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W W W

• Phenix
• www.phenix-online.org
• Xtriage tutorials
• www.phenix-online.org/tutorials
• CCTBX
• cctbx.sf.net

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