CrysAlis Pro Proteins, Large Unit Cells and Difficult Data Sets

Transcription

1 CrysAlis Pro Proteins, Large Unit Cells and Difficult Data Sets Tadeusz Skarzynski Agilent Technologies UK Mathias Meyer Agilent Technologies Poland Oliver Presly Agilent Technologies UK

2 Seminar Layout Overview of Agilent X-ray systems The PX mode of CrysAlis Pro 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 CrysAlis Pro in structure solution 2

3 X-ray Product Configurations Modular Products - Tuned to Application 4-circle Goniometer X-ray Sources CCD Detectors 3

4 Systems for Superior X-ray Data Quality NEW NEW Xcalibur & Gemini SuperNova GV1000 PX Scanner Small Molecule Protein 4

5 SuperNova vs. Rotating Anode Systems Comparison of data collected from the same weakly-diffracting crystal (cross-linked lysozyme, sealed in protective resin) on different X-ray systems Experimental conditions: - Crystal detector distance 90mm - Exposure time 20s - Rotation angle 1º - Number of frames 100 Data processed with CrysAlis Pro and scaled with AIMLESS (CCP4) 5

6 SuperNova vs. Rotating Anode Systems R-merge Resolution 007HF-CCD 007HF-IP SuperNova FRE-CCD Overall HF-CCD 007HF-IP SuperNova FRE-CCD

7 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 ( Å difference) SuperNova as a sealed tube micro-focus system has much lower maintenance requirements and running costs (very low carbon footprint) 7

8 Agilent SuperNova - Fast data collection Lysozyme crystal Total data collection time 76 sec 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. 8

9 Agilent Software: CrysAlis Pro CrysAlis Pro 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 PX SM 9

10 A Perfect Tool from Crystal to Structure! Mounting Screening Strategy Experiment Structure 10

11 Protein Data Collection and Reduction CrysAlis Pro 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 11

12 PX Menu Options 12

13 Crystal Screening experiment name and folder crystal mounting and centering experiment parameters additional parameters 13

14 Crystal Screening We need to evaluate: Resolution Diffraction quality Unit cell 14

15 Protein crystals can have different diffraction properties even if grown from similar conditions Images from Agilent PX Scanner 2.67Å 2.83Å 1.81Å 1.81Å 15

16 Protein crystals can have different diffraction properties even if grown from similar conditions 16

17 Crystal Screening Decision point 17

18 The Strategy of PX Data Collection 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) 18

19 Frame Rotation Angle and Overlaps Per-frame rotation angle typically used is 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 ( deg) for large cell dimensions and to improve data processing using 3-D profiles in CrysAlis Pro. 19

20 CrysAlis Pro Strategy 20

21 CrysAlis Pro Strategy Key Parameters 21

22 CrysAlis Pro Strategy Inspection of Runs 22

23 CrysAlis Pro Strategy Inspection of Runs High kappa position 23

24 CrysAlis Pro Strategy Advanced Options 24

25 CrysAlis Pro Strategy Advanced Options 25

26 CrysAlis Pro Strategy 26

27 CrysAlis Pro Strategy Kappa Angle Control No high kappa positions 27

28 CrysAlis Pro Strategy - Overlaps 28

29 CrysAlis Pro Strategy Long Axes Rotation around principle axis 29

30 CrysAlis Pro Strategy Mosaic Crystals Crystal mosaicity, detector distance and frame rotation angle 30

31 CrysAlis Pro 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 Final data statistics 31

32 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 32

33 Manual Data Reduction Special Parameters 1. reduction of profile mask sizes 2. resolution cutoff 3. bad profile filters 33

34 PX Data Refinalisation (rescaling) Possibility to apply several additional corrections after data reduction Absorption Outlier filters Resolution cut-off Space group change Scaling parameters 34

35 Refinalisation Filters Main filters: R int, image/run number, resolution Used to remove outliers and improve data 35

36 External Frame & File Formats CrysAlis Pro can process frames from most detector manufacturers 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, HKL

37 Synchrotron Data Processing Synchrotron data collected at SLS on Pilatus 2M detector (Dectris) Pixel size 172 x 172 μm 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 CrysAlis Pro, scaled with SCALA Data thanks to Sandro Waltersperger, Swiss Light Source 37

38 Synchrotron Data Processing Data statistics Overall InnerShell OuterShell Low resolution limit High resolution limit Rmerge (within I+/I-) Rmerge (all I+ and I-) Rmeas (within I+/I-) Rmeas (all I+ & I-) Rpim (within I+/I-) Rpim (all I+ & I-) Rmerge in top intensity bin Total number of observations Total number unique Mean((I)/sd(I)) Mn(I) half-set correlation CC(1/2) Completeness Multiplicity Anomalous completeness Anomalous multiplicity DelAnom correlation between half-sets Mid-Slope of Anom Normal Probability Anomalous flag switched ON in input, strong anomalous signal found 38

39 Large Unit Cells β-galactosidase Space group P Cell: a=149.44, b=168.57, c= Å Resolution: 1.8 Å (data collected to 2.0 Å) 39

40 Large Unit Cells β-galactosidase - Merging Statistics N Dmin(A) Rmrg Mn(I/sd) C%poss Mlplct Overall:

41 Difficult Cases Very low resolution High crystal mosaicity Split/multiple crystal Ice rings (insufficient cryoprotection) Ice crystals on the sample Small-molecule crystals in the loop Combination of any of the above Problematic data sets can be significantly improved by careful data reduction and finalisation with CrysAlis Pro 41

42 Twinned Data Processing SuperNova, Atlas detector Data collection time Temperature 1h 19min (77 frames in 1 run) 100K Cell (Å, ) , Twin law 2 components rotated by around axis Symmetry P Overlapping statistics Twin ratio 54:46 30% isolated, 65% partially overlapping, 5% fully overlapping reflections 42

43 NEW Twinning Integration sss Component 1 contribution Component 2 contribution Overall profile Peak de-convolution and simultaneous processing of all components of the multiple reflection to account for the combined peak intensity. 43

44 Twinned Data Processing R-merge 95% of the reflections can be de-convoluted using the new twinned data processing method in CrysAlis Pro Old New inf Overall

45 Two Structures from One Crystal Sample Na-GST-porphyrin Two plate crystals stuck together Approx. crystal sizes: 0.03 x 0.21 x 0.25 mm x 0.18 x 0.45 mm 3 Multiple diffraction pattern 45

46 Two Structures from One Crystal Sample Ewald Pro 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. 46

47 Two Structures from One Crystal Sample Data statistics Form Compl. Mult Rmerge Monoclinic triclinic 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) Thanks to Oluwatoyin A. Asojo, National School of Tropical Medicine, Houston, TX, USA 47

48 Difficult Case Multiple Problems 48

49 Difficult Case Multiple Problems Ice diffraction filtering in Ewald Explorer 49

50 Difficult Case Multiple Problems Twin cell determination in Ewald Explorer 50

51 Use of CrysAlis Pro Data in Structure Determination To use the.mtz file produced by CrysAlis Pro 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 CrysAlis Pro in the near future. 51

52 Use of CrysAlis Pro Data in Structure Determination Preparation of.mtz data file with AIMLESS 52

53 Sulfur-SAD Phasing Human Neuropilin-1 beta Molecular weight : Sulfur atoms: 5 Exposure time (s) 30, 90 Crystal to camera distance (mm) 55.0 Oscillation angle (º) 0.5 Total experiment time 14h 5m Resolution (Å) Space group P Unit-cell parameters (Å) a=62.89, c=87.91 R merge (0.371) Completeness (%) 99.9 (100) Multiplicity 12.4 (5.5) Data from LS 2 lab, Osaka University 53

54 Sulfur-SAD Phasing Human Neuropilin-1 beta Unmerged data from CrysAlis pro Scaling with Scala (CCP4) SAD phasing and electron density modification with ShelxC/D/E 5 Sulfur atoms were found using ShelxD Electron density map after S-SAD phasing Protein model with anomalous differential peaks 54

55 Application Notes Go to - search xrd and choose applications for a full list of application notes 55

56 Software Updates CrysAlis Pro 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, Free multi-user, multi-site license 56

57 Q&A Please type your question into the chat box. Send a direct chat to the Host which is private, or a chat to All Participants which is public 57

58 Thank you for listening! All feedback and comments are welcome 58