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BCM 6200 - Protein crystallography - I Crystal symmetry X-ray diffraction Protein crystallization X-ray sources SAXS Université de Montréal Faculté de Médecine Département de biochimie X-ray sources • The hardware needed for the collection of X-ray diffraction data are 1) X-ray source 2) X-ray detector 3) Crystal rotation instrument to collect the diffracted data • X-ray sources breakdown into three kinds: 1) home sources o Microfocus sealed x-ray tube beam 4 x 108 ph/sec (200m dia) o Rotating Anode Generator ~ 2 x 1010 ph/sec (200m dia) o MetalJet D2 5 x 1010 ph/sec (10m dia) 2) synchrotron X-ray sources – 2nd/3rd generation o CLS CMCF 081D-1 beamline (3rd) 2 x 1012 ph/sec (100 m)2 o APS 14-BM-D beamline (3rd) 2 x 1010 ph/sec (200 m dia) o NSLS X26 beamline (2nd) 5 x 1011 ph/sec (100 um)2 3) X-ray free electron laser (XFEL ) > 4 x 1021 ph/sec in 5–10 fs pulses - improvement in peak brightness ~ ten orders of magnitude over 3rd generation Xray light sources Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 2 Home X-ray sources • X-ray tubes consist of a filament that acts as a cathode. • Electrons are emitted by the glowing cathode and accelerated by several tens of kVs across the vacuum towards the anode, which consists of a metal target made of a characteristic material, usually copper or chromium, for protein crystallography • As the electron beam impacts the anode, the high kinetic energy of the electrons is converted during deceleration into X-rays producing • a continuous spectrum consisting of bremsstrahlung ("braking radiation") and MetalJet (Ga rich alloy • emission lines characteristic for electronic transitions 9.2keV – molten at RT) caused in the anode material. • The characteristic X-ray emissions, which are important for crystallography, have an intensity that is several orders of magnitude higher than the bremsstrahlung. • The specific components of the X-rays emission are cut out from the bremsstrahlung and other emission lines by filters, monochromators or X-ray mirrors, and the resulting monochromatic X-rays are collimated and focused onto the crystals. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 3 Home X-ray sources • The electron energy = e x V (accelerating voltage), where e is the electron charge. • The photon energy is hv = h(c/), where h is Planck's constant, v is the frequency of the radiation, c is the speed of light, and is the wavelength. • Therefore • The sharp peaks in the spectrum are due to electron transitions between inner orbitals in the atoms of the anode material. • The high-energy electrons reaching the anode kick electrons out of low-lying orbitals in the anode atoms. • Electrons from higher orbitals occupy the empty positions and the energy released in this process is emitted as X-ray radiation of specific wavelengths as shown The spectrum from an X-ray tube with a copper anode (8.0 keV). It shows a continuous spectrum and two sharp peaks due to quantized electrons in the copper. I is the energy of the emitted radiation on an arbitrary scale. The wavelength of an in-house source is fixed by the choice of anode target material and is not tunable, as is the case at a synchrotron. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 4 Home X-ray sources • For emission of the characteristic lines in the spectrum, a minimum excitation voltage is required. • For example, for the emission at Cu K line, V should be at least 8 kV. • If a higher voltage is applied, the intensity emitted is stronger with respect to the continuous radiation, up to V / Vmin 4. • The intensity is also proportional to the tube current, as long as the anode is not overloaded. • The heating of the anode caused by the electron beam at the focal spot limits the maximum power of the tube. • This limit can be moved to a higher power loading if anode target rotates at high speed to efficiently distribute and dissipate heat. Université de Montréal Faculté de Médecine Département de biochimie Schematic representation of the atomic energy levels and transitions causing characteristic X-ray wavelengths. K radiation from a transition of the L-shell to the K -shell and K for a transition from the M-shell to the K-shell. Because of the fine structure in the L-shell, K is split into K(1) and K(2). The weighted average value for K(1) and K(2) wavelengths when Cu is the anode material is taken as 1.54178 Å, while K is 1.39217 Å. MetalJet technology replaces the solid anode with a jet of liquid metal that is molten and continuously regenerated. MetalJet sources thus generate more x-rays from a small spot with performance closer to synchrotron-lightsource BCM 6200 5 Synchrotron radiation Insertion device Synchrotron radiation is emitted when a beam of electrons moving close to the speed of light is bent by a powerful magnetic field • • • • Highly intense X-ray beam Wavelength tunable X-ray bunches – picosecond intervals Polarized Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 6 Synchrotron devices Intensity • At a synchrotron facility, bunches of electrons, several GeV in energy, move in a large, carefully steered, closed electron beam loop containing bending elements and linear segments, collectively called the storage ring. • In each section, magnetic devices are inserted - bending magnets in the curved sections, insertion devices called wigglers and undulators in the straight sections - to bend, wiggle or undulate the path of the electrons while they pass around the ring. • Due to the acceleration experienced in the bending magnets or insertion devices, the electrons emit a narrow fan of intense white (polychromatic) radiation ranging from soft UV to hard X-rays over a very tightly defined angle tangential to the ring. • The radiation is 'tunable' by cutting out fine bands (few eV or 10-5 Å wide) of wavelengths appropriate for particular experiments with monochromator crystals that selectively pass the wavelength of choice. From http://internal.physics.uwa.edu.au/~hammond/SyncRes/making-synchrotron-light.htm Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 7 Properties of Synchrotron Radiation Intensity • The main advantage of synchrotron radiation for X-ray diffraction is its high intensity, which can be at least several orders of magnitude greater than for a conventional X-ray tube. • A further advantage is the very low angular divergence of the beam resulting in sharper diffraction spots reducing overlap between diffracted intensities. Tunability • Synchrotron radiation is tunable. Any suitable wavelength in the spectral range can be selected with a monochromator. • This property is essential for routinely solving protein structures by anomalous dispersion. • Wavelengths tuned to 1 A or less reduces absorption and minimizes radiation damage to the protein crystal even at 100 K. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 8 Properties of Synchrotron Radiation Lifetime • The lifetime of a storage ring filling is limited. When the intensity of the radiation has fallen to a certain minimum value, a new injection is required. • Top-off operations is a quasi-continuous injection mode that increases the flux and brightness of a synchrotron source and improves thermal stability of optical components by maintaining a constant current in the storage ring. • Positive charged ions that form accumulate in the electron beam, leading to beam losses. • A beam composed of positrons has a longer lifetime, because positrons repel positive ions created in the residual gas, reducing losses. Time Structure • Synchrotron radiation is produced in flashes by the circulating bunches of charged particles. • Synchrotrons operate in a single or multi bunch mode with a bunch length in the picosecond range. • This allows structural changes in the nanosecond timescale to be observed. Polarization • Synchrotron radiation is highly polarized - fully polarized with the electric vector in the orbit plane (parallel polarization) and a small perpendicular component out of the plane. • Important when applying the correct polarization factor and affects anomalous X-ray scattering of atoms. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 9 Monochromators • Following the Bragg law (2d sin θ = n ), each component wavelength of a polychromatic beam of radiation directed at a single crystal of known orientation and d-spacing will be diffracted at a discrete angle. • Monochromators make use of this fact to selectively remove radiation outside of a tunable energy range, and pass only the radiation of interest • Mirrors reflect polychromatic X-rays at their respective grazing angle. A double monochromator. The rotation axis for changing the wavelength is perpendicular to the plane of the page. This gives a small vertical shift to the exit beam after rotation, but the incoming and exit beams are parallel. Université de Montréal Faculté de Médecine Département de biochimie The white synchrotron light beam impinges onto a polychromator bent crystal, at several different incident angles, reflecting a polychromatic beam. The reflected beam is selected with a specific bandwidth of hundreds of eV and it is horizontally focused at the sample position. Vertical focusing is accomplished by a bendable mirror before the crystal. BCM 6200 10 Detectors • Photographic film is a classical detector for X-ray radiation, but not used much because of the availability of far more sensitive image plates and area detectors. The single advantage of film over area detectors was its superior resolution resulting from its fine grain single photon counters image plates. • CCD (charge coupled device) detectors can collect Xray data at high frequency (seconds exposure times). Their light-sensitive integrated circuit stores and displays the number of X-ray photons collected at each pixel as an electrical charge that can be readout rapidly. They have fast response, low noise, a reasonable dynamic range, and good linearity. A disadvantage is that they operate at very low temperatures (-70° C) to minimize internal noise. • Pilatus - silicon pixel detectors developed at Swiss Light Source. X-rays are converted to an electrical signal by photoelectric effect in silicon and counted directly by a series of cells bonded to the silicon detector, rather than relying on a phosphor. Each pixel has its own amplifier, discriminator (for distinguishing Xrays of the desired energy from noise) and counter circuit; fast response (7 ms readout), no readout noise, superior dynamic range - count rate > 6 × 106 ph/s/pixel, high resolution (pixel) Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 11 Rotation (Oscillation) Instrument • Goniometers are used to rotate crystals. The crystal (and the reciprocal lattice) must rotated through the Ewald sphere to collect all diffracted intensities. • An efficient strategy uses small oscillation (rotation) steps to collect diffracted intensities to avoid overlaps. Data collection strategies should minimize systematic errors such as spot overlaps and maximize data completeness; high redundancy is also an advantage. • Modern oscillation cameras allow rotation of the crystal around one or more axes. The reflections appear in a seemingly disordered arrangement on the detector. • Their position is determined from the following: 1. The crystal orientation. 2. The unit cell parameters in the crystal. 3. The crystal-to-film distance and the wavelength. 4. The film center. • Intelligent data reduction software mentioned previously can recognize (index) the intensity spots, Modern KAPPA Goniometer showing the apply correction factors, and supply the protein incident X-ray beam collimator with beam stop, the CCD detector face, the cold nitrogen jet crystallographer with a final dataset. nozzle, and the protein crystal mounted on the goniometer head Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 12 Radiation Damage • The problem of radiation damage suffered by protein crystals during x-ray structure determination has plagued crystallographers since the early days of the field • Often multiple crystals were required to obtain a complete room temperature data set. • The introduction of macromolecular crystallography cryocooling techniques in the 1990s reduced the rate of radiation damage to the extent that researchers could largely disregard it. • One could normally obtain all of the required data from a single crystal. • Increased lifetime at 100 K relative to room temperature is 70. • However, with the advent of 3rd generation synchrotrons and the provision of undulator fed beamlines, the observation of radiation damage has become commonplace: • Manifested as a reduction in diffracting power and a loss of high-resolution reflections even at cryotemperatures of 100 K. • In addition, the incident x-rays induce specific structural changes in the protein in a well defined sequence, some amino acids being more susceptible than others. • This phenomenon can change the biological information inferred from an x-ray structure, the active sites of enzymes and metalloproteins being particularly affected. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 13 Radiation Damage • X-ray photons cause radical formation, which leads to chemical reactions that gradually destroy crystalline order. • Radicals are produced in the biological macromolecules and the solvent. • Some radicals (e.g., oxygen or hydroxy radicals) diffuse away and exercise their damaging effect at other sites in the crystal. • Radiation damage can be reduced: a. with sensitive X-ray detectors that allow relatively short exposure times, and b. strongly reduced by cooling crystals to cryogenic temperatures (100 and 120 K). • At these temperatures, radicals are still created by the X -ray photons but their diffusion through the crystal is eliminated. • Even at cryotemperatures, specific groups in the protein are nevertheless damaged. a. Disulfide bonds are especially prone to be damaged, leading to bond cleavage. b. Carboxylic acids can be decarboxylated; cysteine, methionine and tyrosine can also suffer. • Data treatment: Extrapolate to zero dose improves value of the measured intensity. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 14 Cryocrystallography and radiation damage (a) A 100K cryocooled protein crystal held in a vitrified cryobuffer loop. (b) A 100K cryocooled protein crystal that has been subjected to data collection at three positions on a synchrotron undulator X-ray beamline and then allowed to warm up in cryobuffer. The release of secondary radiation products on warming has blackened the crystal at the beam spots. Additionally, gas evolved on warming. Garman E (2003) ‘Cool’ crystals: macromolecular cryocrystallography and radiation damage. Curr Op Struct Biol 13(5), 545–551 Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 15 Cryoprotectants How to cryocool : Garman E. (1999) Cool data: quantity AND quality. Acta Crystallogr D Biol Crystallogr. 55(10):1641-53. Garman E (2003) ‘Cool’ crystals: macromolecular cryocrystallography and radiation damage. Curr Op Struct Biol 13(5), 545–551 Bar chart showing the most commonly used cryoprotectant agents. Co-crystallisation refers to conditions that do not require the addition of cryoprotectant agent. Cryobuffers that contain more than one cryoprotectant were grouped into the ‘mixed’ category. Data were compiled from a survey of all papers published in Acta Crystallographica D: 2000, 146; 2001, 175; 2002, 210; Jan–May 2003, 122. MPD, 2-methyl-2,4-pentanediol. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 16 Radiation Damage at 100K The program RADDOSE is widely used to compute the dose absorbed by a macromolecular crystal during an X-ray diffraction experiment. A number of factors affect the absorbed dose, including the incident X-ray flux density, the photon energy and the composition of the macromolecule and of the buffer in the crystal. Radiation damage induces systematic decay in the diffraction pattern. This is evident from the monotonic decrease in the numbers of diffraction spots and resolution estimate. It is clear that the sample decayed significantly after image 300. The blue and red circles show the total number and the number of ‘good’ spots respectively, while the green triangles show the resolution limit. Winter G, McAuley KE. Automated data collection for macromolecular crystallography (2011) Methods Struct. Proteo. 55(1), 81–93 Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 17 Site Specific Radiation Damage at 100K Owen RL, Rudiño-Piñera E, Garman EF. Experimental determination of the radiation dose limit for cryocooled protein crystals. Proc Natl Acad Sci USA. 2006;103(13):4912-7. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 Electron density of the ferritin structures (contoured at 0.2 electrons per Å3) refined from the first eight data sets (Top and Middle), showing the specific structural damage suffered by Glu63, Arg-52 (from a symmetryrelated molecule), and a water molecule. Between each data set, crystals were exposed to controlled periods of unattenuated beam. Bottom shows residue Thr-29 from every other data set. It shows no degradation in the quality of the electron density, similar to the sulfur-containing residue (Cys-48) visible in the top eight maps. The resilience of these residues to high dose illustrates the highly specific nature of radiation damage in proteins. 18 Radiation Safety • High-energy photons of X-rays have a harmful effect on living tissue. Therefore, they must be used with great care, taking all necessary precautions. • Local regulations for the protection of personnel should be obeyed and unauthorized use of X -ray equipment should be forbidden. • A somewhat confusing number of radiation units are in use. • A definition of the important ones is as follows. • The radiation absorbed dose (rad) is a measure for the amount of radiation that corresponds to the energy absorption in a certain medium, such as a tissue. One rad is the dose of radiation corresponding to an energy absorption of 0.01 J/kg medium. • It has been found that the same absorbed dose from different types of radiation does not always have the same harmful effect in biological systems. To convert absorbed dose to dose equivalent, or "rem," where we now consider the biological effects in man, one modifies with a quality factor. For practical scenarios, with low "linear energy transfer" (LET) radiation such as gamma or x rays, 1 rad = 1 rem. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 19 X-ray free electron laser (XFEL) • An unique capability of XFELs is their peak flux or brightness (flux per source size and per emission angle) • An XFEL source emits the same number of X-rays that we can get today in 1 second from a 3rd generation synchrotron source (1012-1013 ph/s) in a single ultra-fast single burst of about 100 femtoseconds. • Hence XFELs allow snapshots to be taken of the motion of atoms, in effect almost freezing their motion, and of chemical processes such as bond breakage. • A second feature of XFELs is the elimination of the need of growing crystals of sufficient size for structure determination. • With 3rd generation synchrotrons, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. • Using femtosecond pulses from a X-ray free-electron laser, structure determination can be undertaken from single-crystal X-ray diffraction ‘snapshots’ collected from a fully hydrated stream of nanocrystals. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 20 How it works: X-ray free electron laser • The key to understanding of how an XFEL works is the free-electron laser instability or self-organization of electrons in a relativistic beam. • This instability takes an electron beam with a random electron position distribution, and changes it into a distribution with electrons regularly spaced at about the x-ray wavelength, producing what could be called a 1-dimensional electron crystal. This creates spikes of intensity. Basic ingredients of a free-electron laser, including an electron gun, an accelerator and an 'undulator' or 'wiggler' that produces a periodic B-field. Bottom: as the electron bunch travels along the undulator or wiggler, microbunches are progressively built. The microbunching is due to the interaction between the travelling electrons and the previously emitted waves. Ribic PR, Margaritondo G (2012) Status and prospects of x-ray free-electron lasers (X-FELs): a simple presentation. J. Phys. D: Appl. Phys. 45 213001 doi:10.1088/0022-3727/45/21/213001 Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 21 Data Collection: X-ray free electron laser Layout of the diffraction experiment. The XFEL beam is focused on the liquid jet, which contains fully hydrated crystals. The diffracted x-rays are collected with two CCD detectors. The inset in the upper-left corner shows a scanning electron microscope (SEM) image of the nozzle, the flowing jet and focusing gas. Technical details from Chapman H N & 87 others (2011). Femtosecond X-ray protein nanocrystallography. Nature, 470(7332), 73-77. • Nanocrystals flow in their buffer solution in a gas-focused, 4-μm-diameter jet at a velocity of 10 m s−1 perpendicular to the pulsed XFEL beam that is focused on the jet. • Two pairs of high-frame-rate pnCCD detectors record low- and high-angle diffraction from single X-ray FEL pulses, at the FEL repetition rate of 30 Hz. • Crystals arrive at random times and orientations in the beam, and the probability of hitting one is proportional to the crystal concentration. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 22 Proof of Concept • Proof of concept with photosystem I, one of the largest membrane protein complexes. • More than 3,000,000 diffraction patterns were collected and a three-dimensional data set was assembled from individual photosystem I nanocrystals (200 nm to 2 μm in size). • The problem of radiation damage mitigated by using pulses briefer than the timescale of most damage processes. • A new approach to structure determination of macromolecules that 1) do not yield crystals of sufficient size for studies using conventional radiation sources or 2) are (a) Diffraction 'snapshot' of a single nanocrystal of the protein particularly sensitive to radiation damage. photosystem I. Some peaks labeled with their Miller indices (hkl). The resolution in the lower detector corner is 8.5 Å. (b) Diffraction data from 15000 nanocrystal patterns merged into a single dataset, displayed on the linear colour scale shown on the right. (c) Region of the 2mFo − DFc electron density map at 1.0σ (purple mesh), calculated from the 70-fs data and (d) obtained from conventional synchrotron diffraction experiments on photosystem I single crystals (for comparison). From Chapman H N & 87 others (2011). Femtosecond X-ray protein nanocrystallography. Nature, 470(7332), 73-77 Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 23 Outrunning radiation damage (a) Lyosozyme exposed to an X-ray pulse with an FWHM of 2 fs, and disintegration followed in time. Atomic positions in the first two structures (before and after the pulse) are practically identical at this pulse length because of an inertial delay in the explosion. (c) Behavior of lyosozyme during an Xray pulse with an FWHM of 50 fs (b) Lysozyme exposed to the same number of photons as in a, but the FWHM of the pulse was 10 fs. Images show the structure at the beginning, in the middle and near the end of the X-ray pulse Université de Montréal Faculté de Médecine Département de biochimie Using a computer simulation, by the end of a XFEL 100-fs pulse each atom of lysozyme was ionized once, on average, and motion of nuclei had begun. Neutze R, Wouts R, van der Spoel D, Weckert E, Hajdu J. (2000) Potential for biomolecular imaging with femtosecond X-ray pulses. Nature. 406(6797):752-7. BCM 6200 24 Experimental setup: Time resolved nanocrystallography • • • • • Experimental setup and intersection of the 3 interacting beams: X-rays, pump laser, and the liquid jet containing the co-crystals. The upper left insert shows the thermal glow of the 2 keV X-rays interacting with the liquid jet. The lower insert shows the scatter from the frequency-doubled Nd:YAG pump laser from the liquid jet. The overlap of the two beams (liquid jet and X-rays) can be seen, with the pump laser intersecting the liquid jet of cocrystals and extending upstream towards the nozzle. The pump laser is extended upstream of the X-ray pulse to compensate for the approximately 130 μm travel of the crystals between the pump and probe pulses for the 10 μs delay time. Aquila & 79 et al. (2012) Time-resolved protein nanocrystallography using an X-ray free electron laser. Opt Express. 20(3): 2706–2716 Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 25 Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation (A) Stereo figure, showing the structure of myoglobin as well as the F(light) – F(dark) difference electron density for the 0.5 ps time delay data, contoured at +3σ (green) and –3σ (red). (B to D) Difference electron density maps (F(light) – F(dark), showing the bound and photodissociated CO, the doming of the heme, the out-of-plane movement of the iron, and the concomitant movement of His93 away from the heme as well as the rotation of His64 and the movement of Phe43, which is displaced in different directions at the two time delays shown (0.5 and 10 ps). The orientation in panel (B) is rotated by 90° with respect to the orientation in panels (C) and (D). Barends TR, Foucar L, Ardevol A, Nass K, Aquila A, Botha S, Doak RB, Falahati K, Hartmann E, Hilpert M, Heinz M, Hoffmann MC, Köfinger J, Koglin JE, Kovacsova G, Liang M, Milathianaki D, Lemke H, Reinstein J, Roome CM, Shoeman RL, Williams GJ, Burghardt I, Hummer G, Boutet S, Schlichting I. Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation. Science. 2015 Sep 10. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 26 Coupling of ultrafast local modes to slower, global motions in CO myoglobin Ultrafast heme modes (0.5 - 3 ps), such as heme doming (①), are coupled to the F-helix through residues contacting the heme such as His93 and His97 (②). These motions are transmitted to other parts of the protein ( ~ 10 ps) such as the CD corner (through the Lys98-Lys42 interaction), ultimately resulting in a coupling with slower normal modes (100 ps) of the protein such as the motions of the E- and F-helices (③). Barends TR, Foucar L, Ardevol A, Nass K, Aquila A, Botha S, Doak RB, Falahati K, Hartmann E, Hilpert M, Heinz M, Hoffmann MC, Köfinger J, Koglin JE, Kovacsova G, Liang M, Milathianaki D, Lemke H, Reinstein J, Roome CM, Shoeman RL, Williams GJ, Burghardt I, Hummer G, Boutet S, Schlichting I. Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation. Science. 2015 Sep 10. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 27 Future enhancements • Data are collected on fully hydrated nanocrystals without cryogenic cooling. • Use of X-ray laser pulses that are so short that only negligible X-ray-induced radiation damage occurs during data collection. • Significant improvements in sample utilization by exploiting higher X-ray repetition rates (greater number of crystal hits) or by slowing the liquid flow. • A viscous LCP microjet consuming approximately two orders of magnitude less sample (membrane proteins) developed • An injection system using microcrystals suspended in a grease matrix, makes it possible to collect complete SFX diffraction data with 100 μl or less of microcrystals. • Further efficiency gains would result from indexing and merging a greater proportion of patterns into the 3D data set, which may be achieved by applying methods for merging continuous diffraction patterns of single molecules or by using ‘post-refinement’ to obtain accurate structure factor estimates from fewer diffraction patterns. • These methods will also remove the twinning ambiguity that exists in our current indexing scheme. • Our method also has potential application to the study of chemical reactions, such as the processes in photosynthesis or enzymatic reactions. 1. Chapman H N & 87 others (2011). Femtosecond X-ray protein nanocrystallography. Nature, 470(7332), 73-77. 2. Neutze et al. Membrane protein structural biology using X-ray free electron lasers. Curr Opin Struct Biol. 2015 Sep 2;33:115-125. Review. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 28 Linac Coherent Light Source (LCLS) at SLAC • LCLS - 38,000 megawatt-hours per year • SPring 8 synchrotron - 137,000 megawatt-hours in FY 2010. • NSLS-II will draw up to 20 megawatt (48,000 megawatthours per year) of power, or as much as needed to power about 300,000 light bulbs or 4,000 homes • Canadian Light Source Facility Electrical Budget: $1.6 million Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 29 Cutting Edge • Coherent diffractive imaging (CDI) is a “lensless” technique for 2D or 3D reconstruction of nanoscale structures including proteins A highly coherent beam of x-rays illuminates an object. The beam scattered by the object produces a diffraction pattern. The recorded diffraction pattern is then used to reconstruct an image via an iterative feedback algorithm. o Here, the objective lens in a typical microscope is replaced with software to reconstruct the real space image from the reciprocal space diffraction pattern. o The advantage in using no lenses is that the final image is aberration–free and so resolution is only diffraction and dose limited (dependent on wavelength, aperture size and exposure). A simple Fourier transform retrieves only the intensity information and so is insufficient for creating an image from the diffraction pattern due to the phase problem. • Current limitation for high intensity coherent X-ray sources is being removed using bright 3rd and 4th generation synchrotrons or XFEL sources Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 30 Ptychography : Coherent Diffractive Imaging Ptychography is an experimental method developed in the 1970s for electron microscopy which consists of measuring multiple diffraction patterns by scanning an extended sample through the coherent X-ray beam while adjacent probe spots overlap significantly in the object plane. Overlap between adjacent illumination positions provides overdetermination in the data (redundancy). The Ptychographical Iterative (Engine) algorithm (PIE) uses the redundant information provided by the overlap of many diffraction patterns (novel oversampling condition) • Advantages are noise tolerance, high speed of convergence and high resolution of reconstructed images. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 31 FIRST HIGH RESOLUTION 3D X-RAY DIFFRACTION MICROSCOPY of a VIRUS Coherent x-ray diffraction patterns were obtained from a single, unstained herpesvirus virion, and then directly inverted to yield quantitative and high contrast electron density maps with a resolution of 22 nm. The quantitative structure of the viral capsid inside the virion was visualized. Schematic layout of the x-ray diffraction microscope. A 20-µm pinhole was used to define the incident xray beam. The virion specimen was positioned at a distance of 1 m from the pinhole. A silicon guard slit with beveled edges was used to eliminate the parasitic scattering from the pinhole. The oversampled diffraction pattern, recorded on a liquidnitrogen-cooled CCD camera, was directly inverted to a high-contrast image using an iterative phase retrieval by the guided hybrid-inputoutput algorithm (GHIO) Song et al. Quantitative imaging of single, unstained viruses with coherent x rays. Phys Rev Lett. 2008 Oct 10;101(15):158101. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 Present resolution (circa 2010) was limited by the coherent x-ray flux, higher resolutions should be achievable by using more brilliant synchrotron radiation sources 32 Ptychography : Protein nanocrystals and 2D-crystals • In an extension of ptychography to 2D-crystal diffraction, a large (~ several μm) 2dcrystal is illuminated at randomly chosen positions with the tightly-focused (100 nm) coherent XFEL beam. • The resulting scattering is a convolution of crystalline Bragg peaks and finite-aperture coherent scattering features. • Each exposure represents a different alignment between the periodic lattice and the illumination function describing the spot, and although it locally destroys the sample, successive exposures from undamaged regions of the sample provide partiallyredundant information. • Giewekemeyer et al. have demonstrated that this multiple exposure method aids in solving the phase problem and hence facilitates the recovery of the projected highresolution structure of a membrane protein. • By repeated scans at different tilt angles of the 2D-crystal, it may be possible to obtain the 3D-protein structure. Giewekemeyer K, Thibault P, Kalbfleisch S, Beerlink A, Kewish CM, Dierolf M, Pfeiffer F, Salditt T. Quantitative biological imaging by ptychographic x-ray diffraction microscopy. Proc Natl Acad Sci U S A. 2010 Jan 12;107(2):529-34. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 33 Speckle and lensless imaging (a) Whereas the scattering of incoherent radiation, e.g. from a synchrotron, yields only the average spacing d of a collection of scatterers (a), the scattering of coherent radiation (with beam diameter a) produces a rich speckle pattern (b), which can be inverted to obtain the exact distribution of scatterers. (b) Van der Veen JF & Pfeiffer F. Coherent x-ray scattering,” J. Phys. Condens. Matter 16, 5003-5030 (2004) The phase-retrieval process is performed in an iterative manner, by making an initial assumption for the phases and repeatedly Fourier and inverse-Fourier transforming between the real and scattering (reciprocal) space representations. Constraints are applied in each iteration cycle. o In reciprocal space, it is required that the calculated scattering intensity agree with the measurements. o In addition, one or more real-space constraints are applied: such as that scattering from the sample is zero outside a delimiting boundary, that the sample’s electron density is non-negative, or that the scattering is concentrated in isolated “atoms”. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 34 Fluctuation X-ray Scattering http://www.camera.lbl.gov/#!fluctuation-scattering/w29pl • Although X-ray crystallography has been successful at determining several thousands of macromolecular structures, it is limited to studying objects which can be formed into crystals. • Furthermore the structure of biological nanomachines may have a different conformation in their natural environment than what is determined through crystallography. • Therefore, as an alternative and complementary technique, structural biologists often gather diffraction patterns from particles in solution. • However, in these so called small- and wide-angle X-ray scattering experiments, the particles rotate during the imaging process, which yields angularly isotropic data and results in a loss of information that often ultimately leads to poorly determined structural hypotheses. • One solution to overcome the limitations of SAXS and WAXS is to perform the imaging process below rotational diffusion times, so that the particles are frozen in position and orientation during imaging. • From these so-called fluctuation X-ray scattering (FXS) experiments, speckle patterns emerge which have angularly varying intensities. • By computing angular correlations of these speckle patterns, one can extract information several orders of magnitude larger than traditional SAXS and WAXS, which allows one to obtain medium-tohigh resolution three-dimensional reconstructions of macromolecular structure. Donatelli JJ, Zwart PH, Sethian JA. Iterative phasing for fluctuation X-ray scattering. Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):10286-91. Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 35 SAXS and WAXS: Fluctuation X-ray Scattering Model of Model of the X-ray projection of the pentameric ligand-gated ion channel (pLGIC). Reconstruction from 3D FXS data using M-TIP. Resolution 4.8Å Malmerberg E, Kerfeld CA, Zwart PH. Operational properties of fluctuation X-ray scattering data. IUCrJ. 2015 Mar 20;2(Pt 3):309-16. Université de Montréal Faculté de Médecine Département de biochimie • X-ray scattering images collected on timescales shorter than rotation diffusion times using a (partially) coherent beam result in a significant increase in information content in the scattered data. • These fluctuation X-ray scattering (FXS) measurements which are typically performed on an X-ray free-electron laser (XFEL) can provide detailed structure of biological molecules beyond what can be obtained with standard X-ray scattering techniques. • In order to understand, use and validate experimental FXS data, the availability of basic data characteristics and operational properties is essential. • Recently, a computational approach has been developed to interpret FXS data and properties that shows: o How FXS data can be used to derived structural models o Its generalization to the Guinier and Porod laws o How to plan experiments and assess the quality of experimental data BCM 6200 36 Free-electron laser instability I. Electrons, propagating through the undulator, interact with the electromagnetic field generated by other electrons. The interaction changes the electron energy, and the change is modulated with the same period of the radiation wavelength, . II. In the undulator magnetic field the trajectory of electrons with larger (smaller) energy is bent less (more). The length of the electron trajectory changes and the electrons within one radiation wavelength tend to get nearer to each other. III. Since electrons are now near to each other within a wavelength, their electromagnetic fields superimpose in phase, and the total field has a larger amplitude. This in turn means that the interaction between the electrons and the electromagnetic field becomes stronger the mechanism for getting them together becomes more effective. The result is that the amplitude of the electromagnetic field grows exponentially. The rate at which it grows is called the gain length, and is the most important parameter characterizing the instability. The exponential growth saturates when all the electrons are well ordered, and sing perfectly in tune! http://www-ssrl.slac.stanford.edu/stohr/xfels.pdf Université de Montréal Faculté de Médecine Département de biochimie BCM 6200 37