SFX and Laue Diffraction Quan Hao School of Biomedical Sciences 12/2/2018

Diffraction before Destruction Sub-micron Crystals Room Temperature Serial Femtosecond Crystallography (SFX)： Diffraction before Destruction Sub-micron Crystals Room Temperature

APS data (7.7 Å) vs. LCLS data (3.3 Å) Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561 (2015). APS data (7.7 Å) vs. LCLS data (3.3 Å) 12/2/2018

Structural basis for selectivity and diversity in angiotensin II receptors Haitao Zhang et al. Nature 544, 327–332 (20 April 2017) 12/2/2018

Zhang at al. Volume 2 | Part 3 | May 2015 | Pages 322–326 A one-dimensional powder diffraction pattern (e.g. from a mixture of zeolites) seen using conventional methods (left) may potentially be analysed as three-dimensional single-crystal patterns using serial crystallography (right). A one-dimensional powder diffraction pattern seen using conventional methods (left) may potentially be analysed as three-dimensional single-crystal patterns using serial crystallography (right). [Hao, Q. (2015). IUCrJ, 2, 307-308. doi:10.1107/S2052252515004017] Zhang at al. Volume 2 | Part 3 | May 2015 | Pages 322–326 Hao, Volume 2 | Part 3 | May 2015 | Pages 307–308

Improve SFX? Challenges: partial reflections; massive number of crystals (~1M) needed; difficult to process. Proposed SwissFEL bandwidth ~ 3% by strong pulse compression (save time and sample by 30-fold comparing to mono mode) 12/2/2018

Laue Diffraction: a stationary crystal during exposure; polychromatic beam; wavelength normalization required. 12/2/2018

Laue Diffraction Large number of spots on a single image. Non-oscillation sample (e.g. in situ plates) Potential time-resolved application Examples: Moffat, Szebenyi & Bilderback (1984), Science, 223, 1423-25. Hadju et al (1987) Nature, 329, 178-181. Bilderback Hoffman & Thiel (1994) Science, 263, 201-203. Genick, …, Moffat & Getzoff (1997) Science, 275, 1471-75. 12/2/2018

Laue v.s. Mono Laue Pro: most full reflections; fewer crystals needed; easier to scale/merge. Con: more sensitive to mosaicity; possible spatial overlap for large unit cell. Mono Pro: less sensitive to mosaicity. Con: partial reflections; more crystals needed; difficult to process. 12/2/2018

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Experimental Procedures MicroMesh (Mitegen) mounts and flash-cooled, or Liquid jet or LCP or in situ plates at room-T Data will be recorded from one crystal at a time using an area detector. For each crystal, one still image will be recorded Data will be indexed, refined and integrated with the program LAUEGEN 12/2/2018

Feasibility study @CHESS-D1 Cornaby,S. , D. M. E. Szebenyi, D. -M Feasibility study @CHESS-D1 Cornaby,S., D. M. E. Szebenyi, D.-M. Smilgies, D. J. Schuller, R. Gillilan, Q. Hao and D. H. Bilderback (2010) Acta Cryst. D66, 2-11. ΔE/E ~30% 12/2/2018

Narrow bandpass Optics Kazmirov et al (2006) J. Synchrotron Rad. 13, 204-210. Multilayer X-ray optics at CHESS: ΔE/E: 0.2%, 2%, 5% and 10% W/B4C: most commonly used multilayer @2% 12/2/2018

CHESS-G1 experiment Multi-layer optic (~1.7% bandpass: 0.975-0.992 Å) One set of slits (~0.1 x 0.1 mm2) Lysozyme crystal (a=b=79, c=38Å, P43212) Room temperature 72 still images; =2.5; 1 s exposure Crystal-detector (Q4) dist. = 188 mm 12/2/2018

Diffraction image 12/2/2018

Data processing Using DENZO/MOSFLM to find crystal orientation Using LAUEGEN* to refine orientation, improve soft limits (lmin,lmax & dmin) and integrate Using LAUENORM to normalize, scale and merge. *Campbell, J. W., Hao, Q., Harding, M. M., Nguti, N. D. & Wilkinson, C. (1998). LAUEGEN version 6.0 and INTLDM. J. Appl. Cryst. 31, 496-502. 12/2/2018

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Data statistics Resolution range: 12.0 – 2.2 Å Unique reflections: 4370 I/(I) = 10.0 Rmerge = 13.4% (~6% between 0.981-0.985Å) Redundancy = 2.0 Completeness = 70.7% 12/2/2018

Structure solution and refinement Straightforward Molecular replacement solution with AMoRe: c.c.=75%, R=31.7% Refinement with REFMAC: R ~ 20%. 12/2/2018

Electron density map (residues 40-45, 50-55, 58-60) 12/2/2018

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Discussion Ideal bandwidth? small unit cells (<100Å), ~10%; large unit cells (>100Å); ~6%. At 1.7% bandwidth, ~1°. 12/2/2018

Proposed feasibility study at SSRF Beamline: a PX beamline with Multilayer-optics Scientific Goal： To determine protein structure using sub-micron crystals Experimental： Laue diffraction; One-shot-per-crystal structure determination Specifications： Photon energy： 7-16 keV Bandwidth：>3% Beamsize：～20μm x 20μm 12/2/2018

Computation – future plan Daresbury Laue Software Suite LAUEGEN: new algorithm to find orientation; multi-core code to process large amount of images. LAUENORM: better curve-fitting for narrow bandwidth. 12/2/2018

Acknowledgements MacCHESS: Qun Liu, Qingqiu Huang, Chris Heaton, Bill Miller, Marian Szebenyi. CHESS: Detlef Smilgies, Joel Brock, Ernie Fontes, Sterling Cornaby, Don Bilderback, Sol Gruner. Daresbury Lab: John Helliwell, John Campbell, Marjorie Harding. Institute of Physics, CAS Haifu Fan, Tao Zhang Institute of Applied Physics, CAS Dong Wang, Haixiao Deng 12/2/2018