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1.	Boutet, S., et al. (författare) High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography 2012 Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 337:6092, s. 362-364 Tidskriftsartikel (refereegranskat)abstract Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.
2.	Arnlund, David, et al. (författare) Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser 2014 Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 11:9, s. 923-926 Tidskriftsartikel (refereegranskat)abstract We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.
3.	Barty, A., et al. (författare) Self-terminating diffraction gates femtosecond X-ray nanocrystallography measurements 2012 Ingår i: Nature Photonics. - 1749-4885 .- 1749-4893. ; 6:1, s. 35-40 Tidskriftsartikel (refereegranskat)abstract X-ray free-electron lasers have enabled new approaches to the structural determination of protein crystals that are too small or radiation-sensitive for conventional analysis1. For sufficiently short pulses, diffraction is collected before significant changes occur to the sample, and it has been predicted that pulses as short as 10 fs may be required to acquire atomic-resolution structural information1, 2, 3, 4. Here, we describe a mechanism unique to ultrafast, ultra-intense X-ray experiments that allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. Instead, the diffracted X-rays are gated by a rapid loss of crystalline periodicity, producing apparent pulse lengths significantly shorter than the duration of the incident pulse. The shortest apparent pulse lengths occur at the highest resolution, and our measurements indicate that current X-ray free-electron laser technology5 should enable structural determination from submicrometre protein crystals with atomic resolution.
4.	Chapman, Henry N, et al. (författare) Femtosecond X-ray protein nanocrystallography. 2011 Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 470:7332, s. 73-7 Tidskriftsartikel (refereegranskat)abstract X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, 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. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200nm to 2μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
5.	Alonso-Mori, Roberto, et al. (författare) Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode 2012 Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:47, s. 19103-19107 Tidskriftsartikel (refereegranskat)abstract The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this probe-before-destroy approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. K beta(1,3) XES spectra of Mn-II and Mn-2(III,IV) complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to > 100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.
6.	Johansson, Linda C, 1983, et al. (författare) Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography. 2013 Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4 Tidskriftsartikel (refereegranskat)abstract Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8Å resolution and determine its serial femtosecond crystallography structure to 3.5Å resolution. Although every microcrystal is exposed to a dose of 33MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.
7.	Kern, Jan, et al. (författare) Room temperature femtosecond X-ray diffraction of photosystem II microcrystals 2012 Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:25, s. 9721-9726 Tidskriftsartikel (refereegranskat)abstract Most of the dioxygen on earth is generated by the oxidation of water by photosystem II (PS II) using light from the sun. This lightdriven, four-photon reaction is catalyzed by the Mn4CaO5 cluster located at the lumenal side of PS II. Various X-ray studies have been carried out at cryogenic temperatures to understand the intermediate steps involved in the water oxidation mechanism. However, the necessity for collecting data at room temperature, especially for studying the transient steps during the O-O bond formation, requires the development of new methodologies. In this paper we report room temperature X-ray diffraction data of PS II microcrystals obtained using ultrashort (<50 fs) 9 keV X-ray pulses from a hard X-ray free electron laser, namely the Linac Coherent Light Source. The results presented here demonstrate that the probe before destroy approach using an X-ray free electron laser works even for the highly-sensitive Mn4CaO5 cluster in PS II at room temperature. We show that these data are comparable to those obtained in synchrotron radiation studies as seen by the similarities in the overall structure of the helices, the protein subunits and the location of the various cofactors. This work is, therefore, an important step toward future studies for resolving the structure of the Mn4CaO5 cluster without any damage at room temperature, and of the reaction intermediates of PS II during O-O bond formation.
8.	Kern, Jan, et al. (författare) Simultaneous Femtosecond X-ray Spectroscopy and Diffraction of Photosystem II at Room Temperature 2013 Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 340:6131, s. 491-495 Tidskriftsartikel (refereegranskat)abstract Intense femtosecond x-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) of microcrystals of photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S-1) and the first illuminated state (S-2) of PS II. Our simultaneous XRD-XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation-sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.
9.	Loh, N. D., et al. (författare) Cryptotomography : Reconstructing 3D Fourier Intensities from Randomly Oriented Single-Shot Diffraction Patterns 2010 Ingår i: Physical Review Letters. - 0031-9007 .- 1079-7114. ; 104:22, s. 225501-1-225501-5 Tidskriftsartikel (refereegranskat)abstract We reconstructed the 3D Fourier intensity distribution of monodisperse prolate nanoparticles using single-shot 2D coherent diffraction patterns collected at DESY's FLASH facility when a bright, coherent, ultrafast x-ray pulse intercepted individual particles of random, unmeasured orientations. This first experimental demonstration of cryptotomography extended the expansion-maximization-compression framework to accommodate unmeasured fluctuations in photon fluence and loss of data due to saturation or background scatter. This work is an important step towards realizing single-shot diffraction imaging of single biomolecules.
10.	Sierra, Raymond G., et al. (författare) Nanoflow electrospinning serial femtosecond crystallography 2012 Ingår i: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 68, s. 1584-1587 Tidskriftsartikel (refereegranskat)abstract An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 mu l min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 mu l min(-1) and diffracted to beyond 4 angstrom resolution, producing 14 000 indexable diffraction patterns, or four per second, from 140 mu g of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.
11.	Aquila, Andrew, et al. (författare) Time-resolved protein nanocrystallography using an X-ray free-electron laser 2012 Ingår i: Optics Express. - 1094-4087. ; 20:3, s. 2706-2716 Tidskriftsartikel (refereegranskat)abstract We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
12.	Bogan, M. J., et al. (författare) Single-shot femtosecond x-ray diffraction from randomly oriented ellipsoidal nanoparticles 2010 Ingår i: Physical Review Special Topics - Accelerators and Beams. - 1098-4402. ; 13:9, s. 094701- Tidskriftsartikel (refereegranskat)abstract Coherent diffractive imaging of single particles using the single-shot "diffract and destroy" approach with an x-ray free electron laser (FEL) was recently demonstrated. A high-resolution low-noise coherent diffraction pattern, representative of the object before it turns into a plasma and explodes, results from the interaction of the FEL with the particle. Iterative phase retrieval algorithms are used to reconstruct two-dimensional projection images of the object from the recorded intensities alone. Here we describe the first single-shot diffraction data set that mimics the data proposed for obtaining 3D structure from identical particles. Ellipsoidal iron oxide nanoparticles (250 nm x 50 nm) were aerosolized and injected through an aerodynamic lens stack into a soft x-ray FEL. Particle orientation was not controlled with this injection method. We observed that, at the instant the x-ray pulse interacts with the particle, a snapshot of the particle's orientation is encoded in the diffraction pattern. The results give credence to one of the technical concepts of imaging individual nanometer and subnanometer-sized objects such as single molecules or larger clusters of molecules using hard x-ray FELs and will be used to help develop robust algorithms for determining particle orientations and 3D structure.
13.	Iwan, Bianca S, et al. (författare) TOF-OFF : A method for determining focal positions in tightly focused free-electron laser experiments by measurement of ejected ions 2011 Ingår i: High Energy Density Physics. - : Elsevier BV. - 1574-1818. ; 7:4, s. 336-342 Tidskriftsartikel (refereegranskat)abstract Pulse intensities greater than 1017 Watt/cm2 were reached at the FLASH soft X-ray laser in Hamburg, Germany, using an off-axis parabolic mirror to focus 15 fs pulses of 5–70 μJ energy at 13.5 nm wavelength to a micron-sized spot. We describe the interaction of such pulses with niobium and vanadium targets and their deuterides. The beam produced craters in the solid targets, and we measured the kinetic energy of ions ejected from these craters. Ions with several keV kinetic energy were observed from craters approaching 5 μm in depth when the sample was at best focus. We also observed the onset of saturation in both ion acceleration and ablation with pulse intensities exceeding 1016 W/cm2, when the highest detected ion energies and the crater depths tend to saturate with increasing intensity. A general difficulty in working with micron and sub-micron focusing optics is finding the exact focus of the beam inside a vacuum chamber. Here we propose a direct method to measure the focal position to a resolution better than the Rayleigh length. The method is based on the correlation between the energies of ejected ions and the physical dimensions of the craters. We find that the focus position can be quickly determined from the ion time-of-flight (TOF) data as the target is scanned through the expected focal region. The method does not require external access to the sample or venting the vacuum chamber. Profile fitting employed to analyze the TOF data can extend resolution beyond the actual scanning step size.
14.	Lomb, Lukas, et al. (författare) Radiation damage in protein serial femtosecond crystallography using an x-ray free-electron laser 2011 Ingår i: Physical Review B. Condensed Matter and Materials Physics. - 1098-0121 .- 1550-235X. ; 84:21, s. 214111-1-214111-6 Tidskriftsartikel (refereegranskat)abstract X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.
15.	Thomas, H., et al. (författare) Explosions of Xenon Clusters in Ultraintense Femtosecond X-Ray Pulses from the LCLS Free Electron Laser 2012 Ingår i: Physical Review Letters. - 0031-9007 .- 1079-7114. ; 108:13 Tidskriftsartikel (refereegranskat)abstract Explosions of large Xe clusters (< N > similar to 11 000) irradiated by femtosecond pulses of 850 eV x-ray photons focused to an intensity of up to 1017 W/cm(2) from the Linac Coherent Light Source were investigated experimentally. Measurements of ion charge-state distributions and energy spectra exhibit strong evidence for the formation of a Xe nanoplasma in the intense x-ray pulse. This x-ray produced Xe nanoplasma is accompanied by a three-body recombination and hydrodynamic expansion. These experimental results appear to be consistent with a model in which a spherically exploding nanoplasma is formed inside the Xe cluster and where the plasma temperature is determined by photoionization heating.
16.	Andreasson, Jakob, et al. (författare) Saturated ablation in metal hydrides and acceleration of protons and deuterons to keV energies with a soft-x-ray laser 2011 Ingår i: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics. - 1539-3755 .- 1550-2376. ; 83:1, s. 016403- Tidskriftsartikel (refereegranskat)abstract Studies of materials under extreme conditions have relevance to a broad area of research, including planetary physics, fusion research, materials science, and structural biology with x-ray lasers. We study such extreme conditions and experimentally probe the interaction between ultrashort soft x-ray pulses and solid targets (metals and their deuterides) at the FLASH free-electron laser where power densities exceeding 1017 W/cm2 were reached. Time-of-flight ion spectrometry and crater analysis were used to characterize the interaction. The results show the onset of saturation in the ablation process at power densities above 1016 W/cm2. This effect can be linked to a transiently induced x-ray transparency in the solid by the femtosecond x-ray pulse at high power densities. The measured kinetic energies of protons and deuterons ejected from the surface reach several keV and concur with predictions from plasma-expansion models. Simulations of the interactions were performed with a nonlocal thermodynamic equilibrium code with radiation transfer. These calculations return critical depths similar to the observed crater depths and capture the transient surface transparency at higher power densities.
17.	Frank, Matthias, et al. (författare) Femtosecond X-ray diffraction from two-dimensional protein crystals 2014 Ingår i: IUCrJ. - 2052-2525. ; 1:2, s. 95-100 Tidskriftsartikel (refereegranskat)abstract X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.
18.	Lee, Ho-Hsien, et al. (författare) Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 2014 Ingår i: IUCrJ. - 2052-2525. ; 1:5, s. 305-317 Tidskriftsartikel (refereegranskat)abstract CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) andthe membrane-proximal region of gp41 (MPR), the transmembrane envelopeprotein ofHuman immunodeficiency virus 1(HIV-1), and has previously beenshown to induce the production of anti-HIV-1 antibodies with antiviralfunctions. To further improve the design of this candidate vaccine, X-raycrystallography experiments were performed to obtain structural informationabout this fusion protein. Several variants of CTB-MPR were designed,constructed and recombinantly expressed inEscherichia coli. The first variantcontained a flexible GPGP linker between CTB and MPR, and yielded crystalsthat diffracted to a resolution of 2.3 A ̊, but only the CTB region was detectedin the electron-density map. A second variant, in which the CTB was directlyattached to MPR, was shown to destabilize pentamer formation. A thirdconstruct containing a polyalanine linker between CTB and MPR proved tostabilize the pentameric form of the protein during purification. The purificationprocedure was shown to produce a homogeneously pure and monodispersesample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered inthe third dimension. Nanocrystals obtained using the same precipitant showedpromising X-ray diffraction to 5 A ̊resolution in femtosecond nanocrystallo-graphy experiments at the Linac Coherent Light Source at the SLAC NationalAccelerator Laboratory. The results demonstrate the utility of femtosecondX-ray crystallography to enable structural analysis based on nano/microcrystalsof a protein for which no macroscopic crystals ordered in three dimensions havebeen observed before.
19.	Milathianaki, D., et al. (författare) Femtosecond Visualization of Lattice Dynamics in Shock-Compressed Matter 2013 Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 342:6155, s. 220-223 Tidskriftsartikel (refereegranskat)abstract The ultrafast evolution of microstructure is key to understanding high-pressure and strain-rate phenomena. However, the visualization of lattice dynamics at scales commensurate with those of atomistic simulations has been challenging. Here, we report femtosecond x-ray diffraction measurements unveiling the response of copper to laser shock-compression at peak normal elastic stresses of similar to 73 gigapascals (GPa) and strain rates of 10(9) per second. We capture the evolution of the lattice from a one-dimensional (1D) elastic to a 3D plastically relaxed state within a few tens of picoseconds, after reaching shear stresses of 18 GPa. Our in situ high-precision measurement of material strength at spatial (<1 micrometer) and temporal (<50 picoseconds) scales provides a direct comparison with multimillion-atom molecular dynamics simulations.
20.	Redecke, Lars, et al. (författare) Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. 2013 Ingår i: Science (New York, N.Y.). - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 339:6116, s. 227-30 Tidskriftsartikel (refereegranskat)abstract The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
21.	Sokolowski-Tinten, Klaus, et al. (författare) Short-pulse Laser Induced Transient Structure Formation and Ablation Studied with Time-resolved Coherent XUV-scattering 2010 Ingår i: INTERNATIONAL SYMPOSIUM ON HIGH POWER LASER ABLATION 2010. - : AIP. ; , s. 373-379 Konferensbidrag (refereegranskat)abstract The structural dynamics of short-pulse laser irradiated surfaces and nano-structures has been studied with nm spatial and ultrafast temporal resolution by means of single-shot coherent XUV-scattering techniques. The experiments allowed us to time-resolve the formation of laser-induced periodic surface structures, and to follow the expansion and disintegration of nano-objects during laser ablation.
22.	Bogan, Michael J., et al. (författare) Aerosol Imaging with a Soft X-Ray Free Electron Laser 2010 Ingår i: Aerosol Science and Technology. - : Informa UK Limited. - 0278-6826 .- 1521-7388. ; 44:3, s. I-VI Tidskriftsartikel (refereegranskat)abstract Lasers have long played a critical role in the advancement of aerosol science. A new regime of ultrafast laser technology has recently be realized, the world's first soft x-ray free electron laser. The Free electron LASer in Hamburg, FLASH, user facility produces a steady source of 10 femtosecond pulses of 7–32 nm x-rays with 1012 photons per pulse. The high brightness, short wavelength, and high repetition rate (> 500 pulses per second) of this laser offers unique capabilities for aerosol characterization. Here we use FLASH to perform the highest resolution imaging of single PM2.5 aerosol particles in flight to date. We resolve to 35 nm the morphology of fibrous and aggregated spherical carbonaceous nanoparticles that existed for less than two milliseconds in vacuum. Our result opens the possibility for high spatial- and time-resolved single particle aerosol dynamics studies, filling a critical technological need in aerosol science.
23.	Hantke, Max F., et al. (författare) High-throughput imaging of heterogeneous cell organelles with an X-ray laser 2014 Ingår i: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 8:12, s. 943-949 Tidskriftsartikel (refereegranskat)abstract We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.
24.	Hattne, Johan, et al. (författare) Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers 2014 Ingår i: Nature Methods. - 1548-7091 .- 1548-7105. ; 11:5, s. 545-548 Tidskriftsartikel (refereegranskat)abstract X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.
25.	Hattne, Johan, et al. (författare) Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers 2014 Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 11:5, s. 545-548 Tidskriftsartikel (refereegranskat)abstract X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.
26.	Hau-Riege, Stefan P., et al. (författare) Sacrificial Tamper Slows Down Sample Explosion in FLASH Diffraction Experiments 2010 Ingår i: Physical Review Letters. - 0031-9007 .- 1079-7114. ; 104:6, s. 064801- Tidskriftsartikel (refereegranskat)abstract Intense and ultrashort x-ray pulses from free-electron lasers open up the possibility for near-atomic resolution imaging without the need for crystallization. Such experiments require high photon fluences and pulses shorter than the time to destroy the sample. We describe results with a new femtosecond pump-probe diffraction technique employing coherent 0.1 keV x rays from the FLASH soft x-ray free-electron laser. We show that the lifetime of a nanostructured sample can be extended to several picoseconds by a tamper layer to dampen and quench the sample explosion, making <1 nm resolution imaging feasible.
27.	Hunter, Mark S, et al. (författare) Fixed-target protein serial microcrystallography with an x-ray free electron laser. 2014 Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4, s. 6026- Tidskriftsartikel (refereegranskat)abstract We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.
28.	Johansson, Linda C, 1983, et al. (författare) Lipidic phase membrane protein serial femtosecond crystallography. 2012 Ingår i: Nature methods. - : Springer Science and Business Media LLC. - 1548-7105 .- 1548-7091. ; 9:3, s. 263-265 Tidskriftsartikel (refereegranskat)abstract X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.
29.	Kern, Jan, et al. (författare) Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy 2014 Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5, s. 4371- Tidskriftsartikel (refereegranskat)abstract The dioxygen we breathe is formed by light-induced oxidation of water in photosystem II. O-2 formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction centre is excited by three single-turnover flashes. Here we present combined X-ray emission spectra and diffraction data of 2-flash (2F) and 3-flash (3F) photosystem II samples, and of a transient 3F' state (250 mu s after the third flash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial O-O bond formation, coupled to Mn reduction, does not yet occur within 250 mu s after the third flash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no significant structural changes at the present resolution of 4.5 angstrom. This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II.
30.	Koopmann, Rudolf, et al. (författare) In vivo protein crystallization opens new routes in structural biology 2012 Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 9:3, s. 259-262 Tidskriftsartikel (refereegranskat)abstract Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo–grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.
31.	Kupitz, Christopher, et al. (författare) Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser 2014 Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 513:7517, s. 261-265 Tidskriftsartikel (refereegranskat)abstract Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
32.	Liu, Wei, et al. (författare) Serial Femtosecond Crystallography of G Protein-Coupled Receptors 2013 Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 342:6165, s. 1521-1524 Tidskriftsartikel (refereegranskat)abstract X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.
33.	Rath, Asawari D., et al. (författare) Explosion dynamics of sucrose nanospheres monitored by time of flight spectrometry and coherent diffractive imaging at the split-and-delay beam line of the FLASH soft X-ray laser 2014 Ingår i: Optics Express. - 1094-4087. ; 22:23, s. 28914-28925 Tidskriftsartikel (refereegranskat)abstract We use a Mach-Zehnder type autocorrelator to split and delay XUV pulses from the FLASH soft X-ray laser for triggering and subsequently probing the explosion of aerosolised sugar balls. FLASH was running at 182 eV photon energy with pulses of 70 fs duration. The delay between the pump-probe pulses was varied between zero and 5 ps, and the pulses were focused to reach peak intensities above 1016 W/cm2 with an off-axis parabola. The direct pulse triggered the explosion of single aerosolised sucrose nano-particles, while the delayed pulse probed the exploding structure. The ejected ions were measured by ion time of flight spectrometry, and the particle sizes were measured by coherent diffractive imaging. The results show that sucrose particles of 560-1000 nm diameter retain their size for about 500 fs following the first exposure. Significant sample expansion happens between 500 fs and 1 ps. We present simulations to support these observations.
34.	Seibert, M. Marvin, et al. (författare) Femtosecond diffractive imaging of biological cells 2010 Ingår i: Journal of Physics B. - : IOP Publishing. - 0953-4075 .- 1361-6455. ; 43:19, s. 194015- Tidskriftsartikel (refereegranskat)abstract In a flash diffraction experiment, a short and extremely intense x-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval by iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al 2006 Nat. Phys. 2 839-43). We report here experiments on biological systems where individual cells were imaged, using single, 10-15 fs soft x-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show that the pulse heated the sample to about 160 000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest that the average displacement of ions and atoms in the hottest surface layers remained below 3 angstrom during the pulse.
35.	Seibert, M. Marvin, et al. (författare) Single mimivirus particles intercepted and imaged with an X-ray laser 2011 Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 470:7332, s. 78-81 Tidskriftsartikel (refereegranskat)abstract X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions(1-4). Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma(1). The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval(2). Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a noncrystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source(5). Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.
36.	Weierstall, Uwe, et al. (författare) Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography 2014 Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5, s. 3309- Tidskriftsartikel (refereegranskat)abstract Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-mu m-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.

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