Serial Crystallography using x-ray Free Electron Lasers Francesco Stellato I.N.F.N. – Sezione di Roma ‘Tor Vergata’ Milan, July 11th 2014 Summary Structural biology and X-rays From synchrotrons to Free Electron Lasers Diffract-and-destroy measurements Serial Crystallography at FELs Sample Preparation and Charcterization Sample delivery Data analysis The Cathepsin B experiment Serial Crystallography at synchrotrons Applications & Future perspectives Structural Biology and X-rays Source Peak Brilliance Perutz & Kendrew myoglobin MacKinnon 1E+36 Potassium channel Franklin, 1E+33 Crick, 1E+30 Watson Bragg & Bragg DNA 1E+27 reflections Hodgkin penicillin, 1E+24 B12 von Laue 1E+21 crystal diffraction 1E+18 1E+15 Röntgen 1E+12 Kornberg 1E+09 RNA Kirz & 1E+06 polymerase Schmahl Jacobsen 1E+03 Microscopy Holography 1E+00 1880 1910 1940 1970 2000 2030 Year Free Electron Lasers (FELs) Radiation is generated by an undulator Electrons are bunched up by interaction with x-rays courtesy: Thomas Tschentscher (XFEL) FELs around the world Hard x-rays Soft x-rays FLASH FLASH LCLS Hamburg, Stanford Germany LINAC Coherent Light Source USA λ > 4.2 nm λ > 0.12 nm SACLA FERMI Trieste Italy Rikken Japan Under construction Synchrotrons and FELs -Similar average brilliance, very different peak brilliance 1012 photons in ~0.05 μm2 FEL Pulse-rate FEL: 100 Hz (so far…) -Different pulse length: 10 -100 fs FEL 10-100 ps sinchrotrons -Short wavelength: Up to 10 keV FELs (first harmonic) Up to 100 keV sinchrotrons Diffraction before destruction Particle injection One pulse, one measure FEL puse Diffraction pattern A detectable signal must be recorded before the sample is destroyed R. Neutze et al, Nature 406 (2000) Coherent x-ray Imaging Diffract and destroy proof-of-principle 1 micron Diffraction pattern SEM picture of a FIB-bed pattern etched on a Si3N4membrane Diffraction pattern Second pulse Reconstructed image at 32 nm resolution FLASH First pulse Chapman et al. Nature Physics (2006) 1 micron Diffraction before destruction The diffract-and-destroy principle can be exported for in principle all synchrotron x-ray techniques - Coherent Imaging - SAXS / WAXS - X-ray Spectroscopies - Crystallography 200 nm 2D reconstruction of a mimi-virus from a single 200 fs LCLS pulse Seibert et al. Nature 470, p.78 (2011) FEL Serial Crystallography 70000 60000 - Measurements of many (103-104) single crystal diffraction patterns 50000 40000 30000 - Indexing - Intensities determination & merging 20000 Electron NMR 1972 1976 X-ray 10000 1980 1984 1988 1992 1996 2000 2004 2008 0 Standard crystallography is the election technique for structural biology - Standard (and non-standard) phasing methods FEL Serial Crystallography Experimental setup •FEL generated x-ray beam •Focusing optics •Sample •Sample injection system •Detector FEL Serial Crystallography Pilot experiment Photosystem I AMO beamline Sun-catcher Membrane protein @ LCLS Upper front CCD beam center 36 proteins 381 cofactors Lower front CCD Resolution at corner = 8.6Å Single shot at LCLS E = 1.8 keV 80 fs pulse 2 mJ pulse energy Chapman et al. Nature 470, (2011) FEL Serial Crystallography Pilot experiment Virtual powder data show that there is no damage up to 70 fs pulses Molecular replacement method is used starting from the known structure LCLS data allowed solving PSI structure at 7 Å resolution (wavelength and geometry limit) The electron density is compatible with the known structure one First FEL based pdb structure Pdb ID: 3PCQ - www.pdb.org Chapman et al. Nature 470, (2011) Nano/micro-crystals Preparation SEM images Standard techniques can be optimized to grow many microand/or nano-crystals: - Hanging droplet (robots) -Batch methods - In vivo crystallization Needleshaped Cathepsin B Nanocrystals A Proteinase K Nanocrystal Nano/micro crystals Screening Several techniques are used to detect nanocrystals: -Optical and electron Microscopy -X-ray diffraction (XRD) (mainly powder) -SONICC Nano/micrco crystals Characterization Several techniques are used to characterize nanocrystals in terms of quality, concentration and size distribution -Dynamic light scattering (DLS) - Optical and electron Microscopy -Differential mobility analysis (DMA) -Nanoparticle tracking analysis (NTA) -X-ray diffraction (XRD) (mainly powder) - SONICC Sample Delivery A good sample delivery system should: -Keep the sample as close as possible to native conditions -Have low background -Deliver a fresh crystal at every FEL pulse -Use as few crystals as possible -Allow pump-probe measurements -Be as stable as possible Systems used so far at FELs: -Gas Dynamic Virtual Nozzle -Lipidic cubic phase nozzle -Aerosol injector -Electrospinning -Fix targets -… Sample Delivery Systems Hitrate (fraction of FEL pulses that hit a sample) is determined by -Sample concentration -Beam diameter -Particle beam diameter -Particle beam stability Examples of hitrate at LCLS Gas Dynamic Virtual Nozzle Gas line Liquid jet 100 m/s 0.5-5 μm diameter 1-10 μl/min 10% hitrate Liquid line Gas line Sample reservoir Gas bottle De Ponte D et al. J. Phys. D 2008 Electrospray/Electrospinning & Drop-on-demand Cone-Jet Mode A drop-on-demand system can be used to generate 20-40 μm diameter droplets An electrospray source can generate small droplets and an associated Differential Mobility Analyzer can sizeselect particles Fix Targets - Sample deposited on thin Si3N4 membranes Ideal for 2D crystallography Frank M. et al., IUCrJ 2014 - Kapton ™ micro-cells Good to keep samples hydrated Zarrine-Asfar A. et al., Acta D 2012 10 μm Data Analysis Flow-chart Diffraction pattern acquisition Hit-finding Only ‘hits’ are processed Background subtraction Sparse patterns: average of many frames Peak finding Peaks are identified in the bkg subtracted patterns Data Analysis Flow-chart Indexing Standard programs (DirAx, MOSFLM, …) called by dedicated softwares (CrystFEL, Cctbx) Intensities The (partial) intensity is evaluated as a locally background subtracted sum of pixels close to the detected (or predicted) peak position Ring-scheme Intensities merging Background Empty region Bragg peak Structure factors White T. et al. J. Appl. Cryst 2012 White T. et al. Acta D 2013 Luci di Sincrotrone CNR – Roma, 22 Aprile 2014 Serial Crystallography – Cathepsin B Cathepsin B Cysteine protease expressed by T.brucei, organism that causes Human African Trypanosomiasis The structure of the protein in the non-native form is known, the glycosylated one not Baculovirus infection of insect cells is commonly used for the expression of proteins requiring post-translational modifications. Luci di Sincrotrone CNR – Roma, 22 Aprile 2014 Serial Crystallography – Cathepsin B Needle-shaped crystals were observed in the cells over-expressing the protein They were purified and concentrated to reach about 109 #/ml 10 ml of concentrated solution were obtained SEM picture of a purified Cathepsin B crystal Serial Crystallography – Cathepsin B Synchrotron data 60s exposure pattern have been collected at DORIS, Hamburg 1010 photons/s in 200x200 μm2 There is a clearly visible ring at 60 Å Faint rings at higher (20-40 Å) resolution. 1s exposure pattern have been collected at SLS, Switzerland 1011 photons/s in 20x20 μm2 Bragg spots are visible up to 8 Å Why such a low resolution? Essentially, because of damage Serial Crystallography – Cathepsin B Measurements at the CXI beamline - LCLS 9.4 keV 40 fs pulse-length 1011 photons/pulse 293,000 hits 175,000 indexed patterns Single crystal diffraction pattern A virtual powder pattern obtained as the sum of thousand single crystals patterns Serial Crystallography – Cathepsin B Projection of the measured intensities on two planes in the reciprocal space Serial Crystallography – Cathepsin B Redecke et al., Science 2013 3D structure of the fully glycosylated protein Serial Crystallography at Synchrotrons Motivations - Room temperature measurements - Time-resolved experiments - Outrun damage (at least partially) Warkentin et al. Acta Cryst. D D67 (2011) Serial Crystallography at Synchrotrons The serial approach can be used at synchrotrons Peak brilliance is lower than FEL‘s one Exposure time must be longer Rotation during the exposure helps integrating the Bragg peak Serial Crystallography at Synchrotrons Beamline P11 @ PETRA III – DESY Hamburg Photon energy: 10 keV Beam size: <10x10 µm2 Flux: 1012 photons/s Detector: PILATUS 6M – 172x172 µm2 pixels Serial Crystallography at Synchrotrons Lysozyme microcrystals grown in batch in high-salt and high viscosity medium Crystal suspension flowing in a thin-walled SAXS capillary at 2.5 l/min Exposure time: 10 ms Serial Crystallography at Synchrotrons > 1,000,000 recorded patterns Hit-finding 150,000 ‘hits’ Indexing 40,000 indexed patterns Bragg spots visible up to 2 Å resolution 2.1 Å Serial Crystallography at Synchrotrons Lysozyme structure solved at 2.1Å resolution by molecular replacement merging intensities from 40,000 single crystal diffraction patterns Pdb ID: 4O34 Stellato F. et al., IUCrJ 2014 Serial Crystallography at Synchrotrons Unit cell parameters are in excellent agreement with known values a = (79.50.3) Å b = (79.40.3) Å c = (38.40.2) Å 10,000 single crystal diffraction patterns would probably be enough Stellato F. et al., IUCrJ 2014 Serial Crystallography at Synchrotrons Cathepsin B – reloaded 500 patterns from Cathepsin B microcrystals at cryogenic temperature Structure solved at 3Å resolution Gati et al., IUCrJ 2014 Luci di Sincrotrone CNR – Roma, 22 Aprile 2014 Applications & Future Perspectives - Time-resolved measurements - Sample delivery optimized for different media - 2D crystallography - Spectroscopies Serial Crystallography Time-resolved Pump-Probe Experiments Changes observed in the putative S3 state in the Photosystem II complex Aquila et al. Optics Express 470 (2011) Kupitz et al. Nature (2014) Serial Crystallography Applications GPCR in Lipidic Cubic Phase Liu et al. Science (2013) Photosyntetic reaction centers in Sponge phase Johanssonn et al. Nature Methods (2012) Scattering & Spectroscpies X-ray emission (XES) X-ray absorption (XAS) Small angle scattering (SAXS) Wide angle scattering (WAXS) Luci di Sincrotrone CNR – Roma, 22 Aprile 2014 Outlook - Less beamtime: higher repetition rate FELs (XFEL) - More sources: brighter sinchrotrons (ESRF, PETRA III) - Less sample: improved sample injection systems - More science: time-resolved experiments on different proteins Higher and higher brilliance will enable approaching the limit of high-resolution single molecule imaging Acknowledgements The Biophysics Group in Tor Vergata Silvia Morante Giancarlo Rossi Velia Minicozzi Francesco Stellato Marco Pascucci Claudia Narcisi Emiliano De Santis biophys.roma2.infn.it CFEL-DESY H. Chapman, J. Schulz, A. Barty, M. Liang, A. Aquila, T. White, D. Deponte, S. Bajt, M. Barthelmess, A. Martin, C. Caleman, K. Nass, F. Stellato, H. Fleckenstein, L. Galli, R. Kirian, K. Beyerlein Arizona State Univeristy J. Spence, P. Fromme, U. Weierstall, B. Doak, M. Hunter, C. Kupitz SLAC M. Bogan, S. Boutet, G. Williams, D. Starodub, R. Sierra, C. Hampton, J. Kryzwinski, C. Bostedt, M. Messerschmidt Uppsala Univeristy J. Hajdu, Nic Timneanu, J. Andreasson, M. Seibert, F. Maia, M. Svenda, T. Ekeberg, J. Andreasson, A. Rocker, O. Jonsson, D. Westphal University of Tübingen, Hamburg and Lübeck C. Betzel, L. Redecke, D. Rehders, K. Cupelli, R. Koopmann, M. Duszenko, T. Stehle Max Planck Heidelberg, LBNL, LLNL European XFEL Massimo Altarellii Thank you for the attention Contacts Francesco Stellato I.N.F.N. Sezione di Roma Tor Vergata Via della Ricerca Scientifica, 1 Tel: 0039 06 7259 4284 [email protected]
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