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Usage of the Kappa goniometer on Proxima1

Usage of the Kappa goniometer on Proxima1. Kappa Meeting @ MAX-lab pierre.legrand@synchrotron-soleil.fr. Overview. General description of the PX1 The 4 main usage of the Kappa + Some illustrating results XO software utilities Conclusion & perspectives. I. General description of proxima1.

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Usage of the Kappa goniometer on Proxima1

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  1. Usage of the Kappa goniometer on Proxima1 Kappa Meeting @ MAX-lab pierre.legrand@synchrotron-soleil.fr 29th Septembre 2009 Kappa Meeting @ MAX-lab

  2. Overview • General description of the PX1 • The 4 main usage of the Kappa + Some illustrating results • XO software utilities • Conclusion & perspectives 29th Septembre 2009 Kappa Meeting @ MAX-lab

  3. I.General description of proxima1 29th Septembre 2009 Kappa Meeting @ MAX-lab

  4. General Layout of PROXIMA 1 Beamline – macromolecular crystallography. Spot size variable 100 – 250 μm Energy 5 – 15 KeV Complementary to micro-focus line (PROXIMA 2) under construction. Primary slits Imager and beam diagnostics Beam shutter Monochromator Cryogenic cooling for monochromator Experimental table + KB Mirrors Robot sample changer, goniostat and detector support. 29th Septembre 2009 Kappa Meeting @ MAX-lab

  5. Experimental layout Mirors: pair Kirpatrick-Baez Goniometre Motorised Table Slits and monitors 29th Septembre 2009 Kappa Meeting @ MAX-lab

  6. Experimental layout CCD Detector ADSC 315r Détecteur Fluorescence 29th Septembre 2009 Kappa Meeting @ MAX-lab

  7. 3 Circle kappa goniometer Sample lighting • Goniometer built by Crystal Logic with tripod goniometric head • Software produced by MSC/Rigaku: cameraman controls: • sample position (XYZ) • backstop (XZ) • sample viewing (focus, zoom, polarisation) • light source (position and intensity) • position of fluorescence detector Roentec mount, translates into place Backstop Beam direction Sample viewing, motorised focus and zoom 29th Septembre 2009 Kappa Meeting @ MAX-lab

  8. Omega Kappa Phi 3 Circle kappa goniometer 29th Septembre 2009 Kappa Meeting @ MAX-lab

  9. Kappa 3 Circle kappa goniometer • Φ, ω axes • DC motor with encoder • Either can be used for data collection • Sphere of confusion • Initial 7.5 µm (now 20 µm) • cylinder of confusion • Φ = 0.6 µm • ω = 1.5 µm 29th Septembre 2009 Kappa Meeting @ MAX-lab

  10. Technical improvements - 1 We have experienced difficulties keeping small crystals aligned (< 20 μm) to the beam even when the beam stability is excellent and the goniometer alignment good. This was investigated as follows. • Improved alignment of gonio – semi automatic protocol using 10 μm W cross hair) makes procedure rapid and simple. • Using this tool to investigate the stability of alignment has identified a slow thermal drift of goniometer with respect to beam (even though mirrors and goniometer are mounted on the same support, which was intended to get rid of this sort of effect!). • 2 “curves superimposed” – fast expansion (metallic components?) and slow expansion (granite?). Note that expansion stabilises after > 36 hours (not shown on curve). • We are working on how to compensate for this (improve air conditioning, remove heat generators from hutch and slow feedback control of goniometer) but “simple solution” (minimise entries into hutch) has greatly improved things. 29th Septembre 2009 Kappa Meeting @ MAX-lab

  11. Technical improvements - 2. • Liquid N2 distribution to be installed at SOLEIL. PROXIMA to be connected to the circuit next moth • He cone developed for reducing signal to noise (long X_ray wavelengths, large unit cells). 29th Septembre 2009 Kappa Meeting @ MAX-lab

  12. Next beamline improvements Major changes to the beamline are planned in the period Sept – December including: • On-axis viewer installation • Pivoting beamstop • Pneumatic “Cryostream” retractor • Cryogenics and support for the robot + ACTOR robot • new cryogenic cooling for the monochromator. • The U20 undulator has been made compatible with 500 mA operation 29th Septembre 2009 Kappa Meeting @ MAX-lab

  13. II.4 main usage of the Kappa with some illustrating results 29th Septembre 2009 Kappa Meeting @ MAX-lab

  14. Exploitation of the kappa goniostat • Mounting & unmouting crytals • Phasing of an structure with weak anomalous signal • Data collection from very large unit cell dimension • Low resolution / Lorenz factor effect 29th Septembre 2009 Kappa Meeting @ MAX-lab

  15. Exploitation of the kappa goniostat • Collect data around φ or ω (selectable from data collection GUI). • Can currently collect -100 < ω < 100 with kappa in the range [–50, +50]. • No limitation with φ • Orienting: collect Bijvoet pairs close together in time or on the same data frame • Disorienting: • Improve completeness • collect high redundancy with different orientation in order to have a better sampling of the absorption effect 29th Septembre 2009 Kappa Meeting @ MAX-lab

  16. A) mounting/unmouting xtals • For mounting and unmounting the frozen crystals users are using the goniometer with: • Kappa: -50 • Omega: 15 29th Septembre 2009 Kappa Meeting @ MAX-lab

  17. B1) Phasing of an structure with weak anomalous signal – Long wavelength • Relatively small protein (131 amino acids, Space group P61 Unit cell 62.45, 62.45, 57.4, 90, 90,120) phased by high redundancy data collected at 2 Ǻwavelength and 2.5 Ǻ resolution using 3 different crystal orientations • κ goniostat at 0, 40, -40 • Phase extension and final model building was done using a 1.7 Ǻ resolution data set (0.98 Ǻ wavelength) recorded from the same crystal. An archea viral protein by S-SAD. Courtesy Adeline Goulet, AFMB, Marseille. 29th Septembre 2009 Kappa Meeting @ MAX-lab

  18. B2) Phasing of an structure with weak anomalous signal – Aligning/combining • The beamline continues to be successful at phasing using anomalous diffraction. Amongst the difficult phasing problems were two more structures solved by S-SAD (Institut Pasteur, Liverpool University), and a structure solved using a rather weak anomalous signal (1 Zn in 280 residues, Univ. Paris Sud). Data from the beamline was used to help solve the structure of a quinolone–DNA cleavage complex of type IIA topoisomerase from S. pneumoniae (Par C + Par E domains, 34 bp DNA duplex + quinolone) by confirming a MR solution using Pt anomalous signal. The problem was difficult because data was relatively low resolution and anisotropic – Nature Structural Biology, 2009. 29th Septembre 2009 Kappa Meeting @ MAX-lab

  19. 29th Septembre 2009 Kappa Meeting @ MAX-lab

  20. C) Data collection from very large unit cell dimension crystals. • Focus the mirrors on the detector for several crystal to detector distances. • ADSC detector in unbinned mode • in conjunction with the choice of orientation using the κ goniometer allowed a group of Marseilles to collect data from a crystal with a difficult unit cell: 78 x 78 x 723 Ǻ space group P 3121, collect up to 2.9 Ǻ resolution Behaviour of beam spot vs distance from focal point using bimorph mirrors. Refocussing routines are available for users, but need substantial maintenance due to hysteresis in the mirror “settings” (particularly for the HFM). 29th Septembre 2009 Kappa Meeting @ MAX-lab

  21. D) Low resolution data collection. • PX1 has been used to collect low resolution data (70% complete to ~ 90 Ǻ) by focussing on the detector, using a small and well aligned beamstop, using the κ goniostat to tilt the crystal and hence modify the Lorentz factor for very low resolution reflections. The resolution ring shown is at ~ 35 Ǻ, and roughly corresponds to the backstop for a “normal” data collection. • Already proven useful (structure solution with starting point cryo-EM model). • The same structure was also solved on PX1 by Se-met MAD (112 Se sites in the asymmetric unit), but due to the difficulty of finding the sites (data collected January 2009) the MR solution preceded this. 29th Septembre 2009 Kappa Meeting @ MAX-lab

  22. III.XO Crystal Orientation utilities A set of python programs to help handling crystal orientations 29th Septembre 2009 Kappa Meeting @ MAX-lab

  23. XO Programs • Basic functionalities for the manipulation of crystal orientation with import/export for XDS, Mosflm and Denzo. • Manipulation include: • Calculation of goniometer settings for realignment XOalign • Conversion XOconvert • Comparison of orientation XOcompare • Handles possible point group permutations 29th Septembre 2009 Kappa Meeting @ MAX-lab

  24. XO Programs • Pure python programs. Only dependency is python >2.1 (linux and osx) • No more Numpy dependency. Numpy has been replaced by the cgtypes classes (cgkit.sf.net) witch is included in the package. • It’s is also jython (java) compatible. • DocTests added. 29th Septembre 2009 Kappa Meeting @ MAX-lab

  25. XO Programs XOconvert • xds2mos, xds2dnz • mos2xds, mos2dnz • dnz2mos, dnz2xds • xds2best xds2mos and dnz2mos write out mosflm input file, so that mosflm can be started automatically to verify predictions xds2dnz write out a Raymond’s strategy input file 29th Septembre 2009 Kappa Meeting @ MAX-lab

  26. XO Programs XOalign • Gonset functionalities + SG permutation + xds import (added to denzo and mosflm) > XOalign.py -V c* -W a* IDXREF.LP > XOalign.py pos2_1_0001.x > XOalign.py -V “[1 1 1]” pos2_1.mat XOcompare • Compare crystal orientations • Tries to find the closest orientation from all the possible Point Group permutations • USAGE: XOcompare.py FILE1 FILE2 29th Septembre 2009 Kappa Meeting @ MAX-lab

  27. The beamline works and we have a good track record of solving structures and sometimes difficult ones weak anomalous signals many site (up to 112 Se) long axes low resolution The cristalLogic Kappa goniometer is an important element of our experimental setup There is room for improvement on what we don’t do well : sample centring small samples optimisation of signal to noise high throughput… Automating data collection to be more efficient is important but not our primary goal – our efforts are targeted towards improving data quality. Conclusion - Status of PX1 29th Septembre 2009 Kappa Meeting @ MAX-lab

  28. Perspectives • Collaboration started with Global Phasing (Peter Keller) to work on the exploitation of the κ goniometer and provide better data collection strategies. • The work of the “Kappa working group” collaboration forms an important part of our future plans. 29th Septembre 2009 Kappa Meeting @ MAX-lab

  29. Team PROXIMA 1 Andy Thompson Pierre Legrand Beatriz Guimarães Patrick Gouhant PROXIMA 2 Bill Shepard Rob Meijers position opened!! Denis Duran Olga Roudenko, Lucile Roussier, Sebastien Lecouster, Majid Ounsy Roger Fourme 29th Septembre 2009 Kappa Meeting @ MAX-lab

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