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CrysAlisPro – Proteins,
Large Unit Cells and
Difficult Data Sets
Mathias Meyer
Agilent Technologies Poland
Oliver Presly
Agilent Technologies UK
Tadeusz Skarzynski
Agilent Technologies UK
Seminar Layout
• Overview of Agilent X-ray systems
• The PX mode of CrysAlisPro
• Crystal screening and data collection strategy for large unit
cells
• Protein data reduction and finalisation
• Advanced features (external detector data, multiple crystals)
• Difficult cases
• Use of data produced by CrysAlisPro in structure solution
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X-ray Product Configurations
Modular Products - Tuned to Application
4-circle Goniometer X-ray Sources
CCD Detectors
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Systems for Superior X-ray Data Quality
4
Small Molecule Protein
Xcalibur & Gemini SuperNova GV1000 PX Scanner
NEW NEW
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SuperNova vs. Rotating Anode Systems
Experimental conditions:
- Crystal detector distance 90mm
- Exposure time 20s
- Rotation angle 1º
- Number of frames 100
Data processed with CrysAlisPro and
scaled with AIMLESS (CCP4)
Comparison of data collected from the same weakly-diffracting crystal
(cross-linked lysozyme, sealed in protective resin) on different X-ray
systems
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SuperNova vs. Rotating Anode Systems
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R-merge
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
2.73.23.74.24.7
007HF-CCD
007HF-IP
SuperNova
FRE-CCD
Resolution 007HF-CCD 007HF-IP SuperNova FRE-CCD
7.31 0.019 0.017 0.009 0.027
5.66 0.023 0.021 0.016 0.028
4.79 0.026 0.029 0.025 0.028
4.22 0.028 0.035 0.033 0.033
3.82 0.037 0.049 0.052 0.047
3.51 0.059 0.097 0.090 0.076
3.27 0.116 0.214 0.187 0.132
3.07 0.234 0.379 0.308 0.229
2.91 0.482 0.778 0.580 0.401
2.76 0.917 1.328 0.994 0.814
Overall 0.037 0.049 0.043 0.043
SuperNova vs. Rotating Anode Systems
• Data collection performance of the SuperNova is very similar to the performance of rotating anode systems equipped with modern CCD detectors and high-flux optics
• The SN performance can be higher than the rotating anode equipped with an image plate due to much lower sensitivity of the IP
• Data resolution is not much different to that obtained on high-end rotating anode systems (0.1-0.2 Å difference)
• SuperNova as a sealed tube micro-focus system has much lower maintenance requirements and running costs (very low carbon footprint)
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Exposure time: 1sec
Readout time: 0.22 sec per image
No. of images: 60
Rotation angle: 1º per image
Data completeness: 96.6 %
Resolution limit: ~1.7 Å
Overall I/σ = 10.8 (last shell = 1.9)
The structure was solved using molecular replacement (MOLREP)
and refined using REFMAC and COOT.
Agilent SuperNova - Fast data collection
Lysozyme crystal
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Total data collection time 76 sec
Agilent Software: CrysAlisPro
CrysAlisPro has been designed to
be as simple to use as possible:
User friendly GUI
Simple workflows
Fully integrated with hardware
Supporting SM and PX
Highly automated......
...but full of manual features for
those who need them
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PX
SM
A Perfect Tool from Crystal to Structure!
Mounting Strategy Structure Experiment Screening
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Protein Data Collection and Reduction
• CrysAlisPro has a dedicated PX mode for running protein
experiments
• Extra features specifically for protein samples
– Simplified work flow
– Dedicated crystal screening
– Modified strategy and finalization parameters
– Import/export of data
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PX Menu Options…
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Crystal Screening…
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experiment
name and
folder crystal
mounting and
centering
experiment
parameters additional
parameters
Resolution
Diffraction
quality
Unit cell
Crystal Screening We need to evaluate:
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1.81Å 2.67Å 2.83Å 1.81Å
Protein crystals can have different diffraction
properties even if grown from similar conditions
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Images from
Agilent PX
Scanner
Protein crystals can have different diffraction
properties even if grown from similar conditions
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Crystal Screening…
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Decision point
We want to achieve:
• best possible resolution
• completeness and redundancy (at least 90% of data and 2-fold multiplicity – more for novel structures)
• optimum frame rotation ranges and avoiding overlaps (crystal orientation, fine slicing)
• highest quality data (at least 1.0 I/sig(I) and R-merge not higher than 50% in the highest
resolution shell)
The Strategy of PX Data Collection
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• Per-frame rotation angle typically used is 0.5 - 1.0 degree but for large cell
dimensions the rotation angle must be smaller.
• For typical crystals with moderate mosaicity (less than 1º):
max rotation = resolution * 57.3 / cell axis (perpendicular to rotation)
• As frame rotation angle increase so do the chances that reflections
passing through the Ewald sphere will overlap each other within a frame.
• The overlaps occur more with high-resolution than with low-resolution
data.
• Use fine-slicing (0.1 – 0.5 deg) for large cell dimensions and to improve
data processing using 3-D profiles in CrysAlisPro.
Frame Rotation Angle and Overlaps
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CrysAlisPro Strategy
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CrysAlisPro Strategy – Key Parameters
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CrysAlisPro Strategy – Inspection of Runs
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CrysAlisPro Strategy – Inspection of Runs
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High kappa position
CrysAlisPro Strategy – Advanced Options
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CrysAlisPro Strategy – Advanced Options
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CrysAlisPro Strategy
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CrysAlisPro Strategy – Kappa Angle Control
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No high kappa positions
CrysAlisPro Strategy - Overlaps
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Rotation around principle axis
CrysAlisPro Strategy – Long Axes
CrysAlisPro Strategy – Mosaic Crystals
Crystal mosaicity, detector distance and frame rotation angle
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CrysAlisPro Strategy – Mosaic Crystals
Crystal mosaicity, detector distance and frame rotation angle
Several trial runs of the Strategy tool indicated:
• Optimum distance 80mm
• Frame rotation 0.3º
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Final data statistics
PX Data Reduction
• Importance of good estimation of data resolution
• Re-processing is always recommended either done by
CrysAlisPro automatically or by the user manually to improve
cell parameters and orientation matrix for data reduction
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Manual Data Reduction – Special Parameters
1. reduction of profile
mask sizes
2. resolution cutoff
3. bad profile filters
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PX Data Refinalisation (rescaling) Possibility to apply several additional corrections after data
reduction
• Absorption
• Outlier filters
• Resolution cut-off
• Space group change
• Scaling parameters
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Refinalisation Filters
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• Main filters: Rint, image/run
number, resolution
• Used to remove outliers and
improve data
External Frame & File Formats
• Simple tools for importing frames
• Esperanto feature for defining new
frame formats
• These features are ideal for
processing synchrotron data
• Data can also be exported in
various file formats like XDS,
Mosflm, HKL2000
CrysAlisPro can process frames from most detector manufacturers
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Synchrotron Data Processing
• Synchrotron data collected at SLS on Pilatus 2M detector (Dectris)
• 4 runs of 10,000 images each (a total of 40,000 images)
• Rotation angle: 0.025º (40 images per degree)
• Data imported and processed fully automatically by CrysAlisPro, scaled with SCALA
Pixel size 172 x 172 μm
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Data thanks to Sandro Waltersperger, Swiss Light Source
Synchrotron Data Processing
Data statistics Overall InnerShell OuterShell
Low resolution limit 39.28 39.28 2.95
High resolution limit 2.80 8.85 2.80
Rmerge (within I+/I-) 0.069 0.046 0.683
Rmerge (all I+ and I-) 0.071 0.048 0.696
Rmeas (within I+/I-) 0.071 0.047 0.704
Rmeas (all I+ & I-) 0.072 0.049 0.707
Rpim (within I+/I-) 0.016 0.010 0.170
Rpim (all I+ & I-) 0.012 0.008 0.123
Rmerge in top intensity bin 0.043 - -
Total number of observations 390160 13998 52089
Total number unique 11270 392 1617
Mean((I)/sd(I)) 33.2 79.7 4.5
Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.974
Completeness 99.9 98.4 100.0
Multiplicity 34.6 35.7 32.2
Anomalous completeness 100.0 99.0 100.0
Anomalous multiplicity 17.9 21.0 16.3
DelAnom correlation between half-sets 0.470 0.578 0.078
Mid-Slope of Anom Normal Probability 1.074 - -
Anomalous flag switched ON in input, strong anomalous signal found
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Large Unit Cells
β-galactosidase
• Space group P212121
• Cell: a=149.44, b=168.57, c=200.63 Å
• Resolution: 1.8 Å (data collected to 2.0 Å)
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Large Unit Cells
β-galactosidase - Merging Statistics
N Dmin(A) Rmrg Mn(I/sd) C%poss Mlplct
1 4.38 0.031 25.9 97.9 3.0
2 3.49 0.041 23.4 100.0 2.9
3 3.05 0.051 14.4 99.9 2.3
4 2.77 0.071 9.6 99.8 2.0
5 2.57 0.099 6.9 99.6 1.8
6 2.42 0.123 4.8 98.9 1.5
7 2.30 0.129 4.0 97.3 1.3
8 2.19 0.158 3.3 95.5 1.3
9 2.11 0.188 2.7 93.8 1.2
10 2.00 0.233 2.2 93.4 1.2
Overall: 0.051 12.0 93.4 1.8
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Difficult Cases
• Very low resolution
• High crystal mosaicity
• Split/multiple crystal
• Ice rings (insufficient cryo-
protection)
• Ice crystals on the sample
• Small-molecule crystals in the
loop
• Combination of any of the
above
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Problematic data sets can be significantly improved by careful data
reduction and finalisation with CrysAlisPro
Twinned Data Processing
SuperNova, Atlas detector
Data collection
time
1h 19min
(77 frames in 1 run)
Temperature 100K
Cell (Å, °) 45.4 51.2 80.9, 90 90 90
Twin law
2 components rotated by -
138.6° around -0.35 0.84 -
0.41 axis
Symmetry P212121
Overlapping
statistics
Twin ratio 54:46
30% isolated,
65% partially overlapping,
5% fully overlapping
reflections
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sss
NEW Twinning Integration
Component 1 contribution Component 2 contribution
Overall profile
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Peak de-convolution and
simultaneous processing
of all components of the
multiple reflection to
account for the combined
peak intensity.
Twinned Data Processing
95% of the reflections can be de-convoluted using the
new twinned data processing method in CrysAlisPro
R-merge
Old New
inf-4.50 0.043 0.03
4.50-3.56 0.062 0.043
3.56-3.12 0.095 0.072
3.12-2.84 0.137 0.095
2.84-2.65 0.182 0.116
2.65-2.49 0.223 0.136
2.49-2.37 0.266 0.167
2.37-2.27 0.31 0.207
2.27-2.18 0.387 0.244
2.18-2.10 0.406 0.272
Overall 0.129 0.089
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Two Structures from One Crystal Sample…
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Na-GST-porphyrin
• Two plate crystals stuck together
• Approx. crystal sizes:
0.03 x 0.21 x 0.25 mm3
0.03 x 0.18 x 0.45 mm3
Multiple diffraction pattern
Two Structures from One Crystal Sample…
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EwaldPro Screenshots
Triclinic form in white, monoclinic form in red. View looking down b* on left, c*
on right. The two domains appear to line up along c*, which is the crystal
stacking direction.
Two Structures from One Crystal Sample…
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The crystal structures of the
monoclinic and triclinic forms
were solved by molecular
replacement in PHASER and
refined with REFMAC to 2.7Å.
For the monoclinic form,
R = 0.23 (Rfree = 0.28)
For the triclinic form,
R = 0.22 (Rfree = 0.28)
Data statistics
Form Compl. Mult Rmerge
Monoclinic 96.1 2.6 0.093
triclinic 84.8 1.5 0.072
Thanks to Oluwatoyin A. Asojo, National School of Tropical Medicine, Houston, TX, USA
Difficult Case – Multiple Problems
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Difficult Case – Multiple Problems
Ice diffraction filtering in Ewald Explorer
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Difficult Case – Multiple Problems
Twin cell determination in Ewald Explorer
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Use of CrysAlisPro Data in Structure Determination
To use the .mtz file produced by CrysAlisPro for structure determination it is
recommended to run CCP4 program AIMLESS to make the data file
compatible with CCP4 and PHENIX conventions. This process will be
automatically carried out by CrysAlisPro in the near future.
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Use of CrysAlisPro Data in Structure Determination
Preparation of .mtz data file with AIMLESS
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Exposure time (s) 30, 90
Crystal to camera distance
(mm) 55.0
Oscillation angle (º) 0.5
Total experiment time 14h 5m
Resolution (Å) 20.8-1.50
Space group P41212
Unit-cell parameters (Å) a=62.89, c=87.91
Rmerge 0.051 (0.371)
Completeness (%) 99.9 (100)
Multiplicity 12.4 (5.5)
Molecular weight : 17533
Sulfur atoms: 5
Sulfur-SAD Phasing
Human Neuropilin-1 beta
Data from LS2 lab, Osaka University
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• Unmerged data from CrysAlispro
• Scaling with Scala (CCP4)
• SAD phasing and electron density
modification with ShelxC/D/E
Electron density map after S-SAD phasing Protein model with anomalous differential peaks
5 Sulfur atoms were found
using ShelxD
Sulfur-SAD Phasing
Human Neuropilin-1 beta
11/11/2013 54
Application Notes
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Go to www.agilent.com/chem/library - search xrd and choose ‘applications’
for a full list of application notes
Software Updates
CrysAlisPro is frequently
updated with fixes for known
problems
New features are introduced
in annual major updates
All updates are Free and
available from our user forum,
www.agilentxrdforum.com
Free multi-user, multi-site
license
11/11/2013 56
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