Serial Crystallography using x-ray Free Electron Lasers Francesco - PowerPoint PPT Presentation

Serial Crystallography using x-ray Free Electron Lasers Francesco Stellato I.N.F.N. – Sezione di Roma ‘Tor Vergata’ Milan, July 11 th 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 Perutz & Kendrew myoglobin MacKinnon 1E+36 Potassium 1E+33 channel Franklin, Source Peak Brilliance Crick, 1E+30 Watson Bragg & Bragg 1E+27 DNA reflections Hodgkin 1E+24 penicillin, B12 von Laue 1E+21 crystal diffraction 1E+18 1E+15 Röntgen 1E+12 1E+09 Kornberg RNA 1E+06 Kirz & polymerase Schmahl 1E+03 Jacobsen 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 Soft x-rays Hard x-rays FLASH FLASH LINAC Coherent Light LCLS Source Hamburg, Stanford Under construction Germany USA λ > 4.2 nm λ > 0.12 nm SACLA FERMI Rikken Trieste Japan Italy

Synchrotrons and FELs - Similar average brilliance, very different peak brilliance 10 12 photons in ~0.05 μ m 2 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, FEL puse one measure 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 Diffraction pattern Diffraction pattern 1 micron SEM picture of a FIB-bed pattern Second pulse etched on a Si 3 N 4 membrane Reconstructed image at 32 nm resolution FLASH First pulse 1 micron Chapman et al. Nature Physics (2006)

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 200 nm 2D reconstruction of a mimi-virus from a single - Crystallography 200 fs LCLS pulse Seibert et al. Nature 470 , p.78 (2011)

FEL Serial Crystallography 70000 - Measurements of many (10 3 -10 4 ) single crystal diffraction patterns 60000 50000 - Indexing 40000 30000 - Intensities determination & merging 20000 - Standard (and non-standard) 10000 Electron phasing methods NMR 0 1972 1976 1980 1984 1988 1992 1996 2000 2004 2008 X-ray Standard crystallography is the election technique for structural biology

FEL Serial Crystallography Experimental setup • FEL generated x-ray beam • Focusing optics • Sample • Sample injection system • Detector

FEL Serial Crystallography Pilot experiment Upper Photosystem I AMO beamline front CCD Sun-catcher Membrane protein @ LCLS 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 Chapman et al. Nature 470 , (2011) Pdb ID: 3PCQ - www.pdb.org

Nano/micro-crystals Preparation SEM images Standard techniques can be Needle- shaped optimized to grow many micro- Cathepsin B and/or nano-crystals: Nanocrystals - Hanging droplet (robots) A Proteinase K -Batch methods Nanocrystal - In vivo crystallization

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 Systems used so far at FELs: should: -Gas Dynamic Virtual Nozzle -Keep the sample as close as possible -Lipidic cubic phase nozzle to native conditions -Aerosol injector -Have low background -Electrospinning -Deliver a fresh crystal at every FEL -Fix targets pulse - … -Use as few crystals as possible -Allow pump-probe measurements -Be as stable as possible

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 100 m/s 0.5-5 μ m diameter 1-10 μ l/min 10% hitrate Gas line Liquid jet Liquid line Gas line Gas bottle Sample reservoir De Ponte D et al. J. Phys. D 2008

Electrospray/Electrospinning & Drop-on-demand An electrospray source can generate small droplets and an associated Differential Mobility Analyzer can size- Cone-Jet Mode select particles A drop-on-demand system can be used to generate 20-40 μ m diameter droplets

Fix Targets - Sample deposited on thin Si 3 N 4 membranes Ideal for 2D crystallography Frank M. et al. , IUCrJ 2014 - Kapton ™ micro -cells Good to keep samples hydrated 10 μ m Zarrine-Asfar A. et al. , Acta D 2012

Data Analysis Flow-chart Diffraction pattern acquisition Hit-finding Only ‘hits’ are processed Sparse patterns: average of many frames Background subtraction Peak finding Peaks are identified in the bkg subtracted patterns

Data Analysis Flow-chart Standard programs (DirAx , MOSFLM, …) called by Indexing dedicated softwares ( CrystFEL, Cctbx ) The (partial) intensity is evaluated as a locally Intensities background subtracted sum of pixels close to the detected (or predicted) peak position Ring-scheme Intensities merging Background Empty region Bragg peak White T. et al. J. Appl. Cryst 2012 Structure factors White T. et al. Acta D 2013

Luci di Sincrotrone CNR – Roma, 22 Aprile 2014 Serial Crystallography – Cathepsin B Cathepsin B The structure of the protein in the Cysteine protease expressed by non-native form is known, the T.brucei, organism that causes glycosylated one not Human African Trypanosomiasis 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 SEM picture of a purified Cathepsin B crystal Needle-shaped crystals were observed in the cells over-expressing the protein They were purified and concentrated to reach about 10 9 #/ml  10 ml of concentrated solution were obtained

Serial Crystallography – Cathepsin B Synchrotron data 60s exposure pattern have been collected at DORIS, Hamburg 10 10 photons/s in 200x200 μ m 2 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 10 11 photons/s in 20x20 μ m 2 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  10 11 photons/pulse 293,000 hits 175,000 indexed patterns A virtual powder pattern Single crystal diffraction 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 µm 2 10 12 photons/s Flux: Detector: PILATUS 6M – 172x172 µm 2 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 Bragg spots visible up to  2 Å  Hit-finding resolution   150,000 ‘ hits ’ 2.1 Å  Indexing  40,000 indexed patterns