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

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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

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