Usage of the Kappa goniometer on Proxima 1

  • Slides: 29
Download presentation
Usage of the Kappa goniometer on Proxima 1 Kappa Meeting @ MAX-lab pierre. legrand@synchrotron-soleil.

Usage of the Kappa goniometer on Proxima 1 Kappa Meeting @ MAX-lab pierre. legrand@synchrotron-soleil. fr

Overview I. II. General description of the PX 1 The 4 main usage of

Overview I. II. General description of the PX 1 The 4 main usage of the Kappa + Some illustrating results III. XO software utilities IV. Conclusion & perspectives

I. General description of proxima 1

I. General description of proxima 1

General Layout of PROXIMA 1 Beamline – macromolecular crystallography. Spot size variable 100 –

General Layout of PROXIMA 1 Beamline – macromolecular crystallography. Spot size variable 100 – 250 μm Primary slits Energy 5 – 15 Ke. V Complementary to micro-focus line (PROXIMA 2) under construction. Beam shutter Imager and beam diagnostics Monochromator Experimental table + KB Mirrors Robot sample changer, goniostat and detector support. Cryogenic cooling for monochro mator

Experimental layout Mirors: pair Kirpatrick. Baez Goniometre Motorised Table Slits and monitors

Experimental layout Mirors: pair Kirpatrick. Baez Goniometre Motorised Table Slits and monitors

Experimental layout CCD Detector ADSC 315 r Détecteur Fluorescence

Experimental layout CCD Detector ADSC 315 r Détecteur Fluorescence

3 Circle kappa goniometer Sample lighting • Goniometer built by Crystal Logic with tripod

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

3 Circle kappa goniometer Omega Kappa Phi

3 Circle kappa goniometer Omega Kappa Phi

3 Circle kappa goniometer • Φ, ω axes Kappa – DC motor with encoder

3 Circle kappa goniometer • Φ, ω axes Kappa – 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

Technical improvements - 1 We have experienced difficulties keeping small crystals aligned (< 20

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.

Technical improvements - 2. • Liquid N 2 distribution to be installed at SOLEIL.

Technical improvements - 2. • Liquid N 2 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).

Next beamline improvements Major changes to the beamline are planned in the period Sept

Next beamline improvements Major changes to the beamline are planned in the period Sept – December including: o On-axis viewer installation o Pivoting beamstop o Pneumatic “Cryostream” retractor o Cryogenics and support for the robot + ACTOR robot ü new cryogenic cooling for the monochromator. ü The U 20 undulator has been made compatible with 500 m. A operation

II. 4 main usage of the Kappa with some illustrating results

II. 4 main usage of the Kappa with some illustrating results

Exploitation of the kappa goniostat A. Mounting & unmouting crytals B. Phasing of an

Exploitation of the kappa goniostat A. Mounting & unmouting crytals B. Phasing of an structure with weak anomalous signal C. Data collection from very large unit cell dimension D. Low resolution / Lorenz factor effect

Exploitation of the kappa goniostat • Collect data around φ or ω (selectable from

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

A) mounting/unmouting xtals • For mounting and unmounting the frozen crystals users are using

A) mounting/unmouting xtals • For mounting and unmounting the frozen crystals users are using the goniometer with: – Kappa: – Omega: -50 15

B 1) Phasing of an structure with weak anomalous signal – Long wavelength •

B 1) Phasing of an structure with weak anomalous signal – Long wavelength • Relatively small protein (131 amino acids, Space group P 61 Unit cell 62. 45, 57. 4, 90, 120) phased by high redundancy data collected at 2 Ǻ wavelength 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 An archea viral protein by S-SAD. same crystal. Courtesy Adeline Goulet, AFMB, Marseille.

B 2) Phasing of an structure with weak anomalous signal – Aligning/combining • The

B 2) 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.

C) Data collection from very large unit cell dimension crystals. 1. Focus the mirrors

C) Data collection from very large unit cell dimension crystals. 1. Focus the mirrors on the detector for several crystal to detector distances. 2. ADSC detector in unbinned mode 3. 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 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).

D) Low resolution data collection. • • • PX 1 has been used to

D) Low resolution data collection. • • • PX 1 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 PX 1 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.

III. XO Crystal Orientation utilities A set of python programs to help handling crystal

III. XO Crystal Orientation utilities A set of python programs to help handling crystal orientations

XO Programs • Basic functionalities for the manipulation of crystal orientation with import/export for

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

XO Programs • Pure python programs. Only dependency is python >2. 1 (linux and

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. • Doc. Tests added.

XO Programs XOconvert – xds 2 mos, xds 2 dnz – mos 2 xds,

XO Programs XOconvert – xds 2 mos, xds 2 dnz – mos 2 xds, mos 2 dnz – dnz 2 mos, dnz 2 xds – xds 2 best xds 2 mos and dnz 2 mos write out mosflm input file, so that mosflm can be started automatically to verify predictions xds 2 dnz write out a Raymond’s strategy input file

XO Programs XOalign – Gonset functionalities + SG permutation + xds import (added to

XO Programs XOalign – Gonset functionalities + SG permutation + xds import (added to denzo and mosflm) > XOalign. py -V c* -W a* IDXREF. LP > XOalign. py pos 2_1_0001. x > XOalign. py -V “[1 1 1]” pos 2_1. mat XOcompare – Compare crystal orientations – Tries to find the closest orientation from all the possible Point Group permutations – USAGE: XOcompare. py FILE 1 FILE 2

Conclusion - Status of PX 1 • The beamline works and we have a

Conclusion - Status of PX 1 • 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 cristal. Logic 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.

Perspectives • Collaboration started with Global Phasing (Peter Keller) to work on the exploitation

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.

Team PROXIMA 1 PROXIMA 2 Andy Thompson Bill Shepard Pierre Legrand Rob Meijers Beatriz

Team PROXIMA 1 PROXIMA 2 Andy Thompson Bill Shepard Pierre Legrand Rob Meijers Beatriz Guimarães position opened!! Patrick Gouhant Denis Duran Olga Roudenko, Lucile Roussier, Sebastien Lecouster, Majid Ounsy Roger Fourme