| 1. |
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| 2. |
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| 3. |
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| 4. |
- Fuller, Franklin D, et al.
(författare)
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Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers
- 2017
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Ingår i: Nature Methods. - : Macmillan Publishers Ltd.. - 1548-7091 .- 1548-7105. ; 14, s. 443-449
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Tidskriftsartikel (refereegranskat)abstract
- X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.
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| 5. |
- Laksmono, Hartawan, et al.
(författare)
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Anomalous Behavior of the Homogeneous Ice Nucleation Rate in No-Man's Land
- 2015
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Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 6:14, s. 2826-2832
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Tidskriftsartikel (refereegranskat)abstract
- We present an analysis of ice nucleation kinetics from near-ambient pressure water as temperature decreases below the homogeneous limit T-H by cooling micrometer-sized droplets (microdroplets) evaporatively at 10(3)-10(4) K/s and probing the structure ultrafast using femtosecond pulses from the Linac Coherent Light Source (LCLS) free-electron X-ray laser. Below 232 K, we observed a slower nucleation rate increase with decreasing temperature than anticipated from previous measurements, which we suggest is due to the rapid decrease in water's diffusivity. This is consistent with earlier findings that microdroplets do not crystallize at <227 K, but vitrify at cooling rates of 10(6)-10(7) K/s. We also hypothesize that the slower increase in the nucleation rate is connected with the proposed fragile-to-strong transition anomaly in water.
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| 6. |
- Li, Haoyuan, et al.
(författare)
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Diffraction data from aerosolized Coliphage PR772 virus particles imaged with the Linac Coherent Light Source
- 2020
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Ingår i: Scientific Data. - : NATURE RESEARCH. - 2052-4463. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited. The diffraction data were collected at the Atomic, Molecular and Optical Science Instrument (AMO) of the LCLS in 4 experimental beam times during a period of four years. The photon energy was either 1.2 or 1.7keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3 mu m x 1.7 mu m full width at half maximum (FWHM). The X-ray laser pulses captured the particles in random orientations. The data offer insight into aerosolised virus particles in the gas phase, contain information relevant to improving experimental parameters, and provide a basis for developing algorithms for image analysis and reconstruction.
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| 7. |
- Munke, Anna, et al.
(författare)
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Data Descriptor : Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source
- 2016
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Ingår i: Scientific Data. - : Nature Publishing Group. - 2052-4463. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a wellcharacterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 mu m diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 angstrom ngstrom were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
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| 8. |
- Schreck, Simon, et al.
(författare)
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Reabsorption of Soft X-Ray Emission at High X-Ray Free-Electron Laser Fluences
- 2014
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Ingår i: Physical Review Letters. - 0031-9007 .- 1079-7114. ; 113:15, s. 153002-
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Tidskriftsartikel (refereegranskat)abstract
- We report on oxygen K-edge soft x-ray emission spectroscopy from a liquid water jet at the Linac Coherent Light Source. We observe significant changes in the spectral content when tuning over a wide range of incident x-ray fluences. In addition the total emission yield decreases at high fluences. These modifications result from reabsorption of x-ray emission by valence-excited molecules generated by the Auger cascade. Our observations have major implications for future x-ray emission studies at intense x-ray sources. We highlight the importance of the x-ray pulse length with respect to the core-hole lifetime.
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| 9. |
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| 10. |
- Sellberg, Jonas A., et al.
(författare)
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X-ray emission spectroscopy of bulk liquid water in no-man's land
- 2015
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 142:4
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Tidskriftsartikel (refereegranskat)abstract
- The structure of bulk liquid water was recently probed by x-ray scattering below the temperature limit of homogeneous nucleation (T-H) of similar to 232 K [J. A. Sellberg et al., Nature 510, 381-384 (2014)]. Here, we utilize a similar approach to study the structure of bulk liquid water below T-H using oxygen K-edge x-ray emission spectroscopy (XES). Based on previous XES experiments [T. Tokushima et al., Chem. Phys. Lett. 460, 387-400 (2008)] at higher temperatures, we expected the ratio of the 1b(1)' and 1b(1) peaks associated with the lone-pair orbital in water to change strongly upon deep supercooling as the coordination of the hydrogen (H-) bonds becomes tetrahedral. In contrast, we observed only minor changes in the lone-pair spectral region, challenging an interpretation in terms of two interconverting species. A number of alternative hypotheses to explain the results are put forward and discussed. Although the spectra can be explained by various contributions from these hypotheses, we here emphasize the interpretation that the line shape of each component changes dramatically when approaching lower temperatures, where, in particular, the peak assigned to the proposed disordered component would become more symmetrical as vibrational interference becomes more important.
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| 11. |
- Young, Iris D., et al.
(författare)
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Structure of photosystem II and substrate binding at room temperature
- 2016
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Ingår i: Nature. - : Macmillan Publishers Ltd.. - 0028-0836 .- 1476-4687. ; 540:7633, s. 453-457
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Tidskriftsartikel (refereegranskat)abstract
- Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution2,3. A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site4–6. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL7 provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10–13. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.
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| 12. |
- Aleksich, Mariya, et al.
(författare)
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XFEL Microcrystallography of Self-Assembling Silver n-Alkanethiolates
- 2023
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Ingår i: Journal of the American Chemical Society. - 0002-7863. ; 145:31, s. 17042-17055
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Tidskriftsartikel (refereegranskat)abstract
- New synthetic hybrid materials and their increasing complexity have placed growing demands on crystal growth for single-crystal X-ray diffraction analysis. Unfortunately, not all chemical systems are conducive to the isolation of single crystals for traditional characterization. Here, small-molecule serial femtosecond crystallography (smSFX) at atomic resolution (0.833 Å) is employed to characterize microcrystalline silver n-alkanethiolates with various alkyl chain lengths at X-ray free electron laser facilities, resolving long-standing controversies regarding the atomic connectivity and odd-even effects of layer stacking. smSFX provides high-quality crystal structures directly from the powder of the true unknowns, a capability that is particularly useful for systems having notoriously small or defective crystals. We present crystal structures of silver n-butanethiolate (C4), silver n-hexanethiolate (C6), and silver n-nonanethiolate (C9). We show that an odd-even effect originates from the orientation of the terminal methyl group and its role in packing efficiency. We also propose a secondary odd-even effect involving multiple mosaic blocks in the crystals containing even-numbered chains, identified by selected-area electron diffraction measurements. We conclude with a discussion of the merits of the synthetic preparation for the preparation of microdiffraction specimens and compare the long-range order in these crystals to that of self-assembled monolayers.
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| 13. |
- Alonso-Mori, Roberto, et al.
(författare)
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Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode
- 2012
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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
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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.
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| 14. |
- Chapman, Henry N, et al.
(författare)
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Femtosecond X-ray protein nanocrystallography.
- 2011
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 470:7332, s. 73-7
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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.
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| 15. |
- Dao, E. Han, et al.
(författare)
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Structure of the 30S ribosomal decoding complex at ambient temperature
- 2018
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Ingår i: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 24:12, s. 1667-1676
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Tidskriftsartikel (refereegranskat)abstract
- The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Therm us thermophilus 30S ribosomal subunit decoding complex to 3.45 angstrom resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.
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| 16. |
- Ekeberg, Tomas, et al.
(författare)
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Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser
- 2016
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Ingår i: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms.
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| 17. |
- Hattne, Johan, et al.
(författare)
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Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers
- 2014
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Ingår i: Nature Methods. - 1548-7091 .- 1548-7105. ; 11:5, s. 545-548
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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.
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| 18. |
- Hattne, Johan, et al.
(författare)
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Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers
- 2014
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 11:5, s. 545-548
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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.
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| 19. |
- Johansson, Linda C, 1983, et al.
(författare)
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Lipidic phase membrane protein serial femtosecond crystallography.
- 2012
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Ingår i: Nature methods. - : Springer Science and Business Media LLC. - 1548-7105 .- 1548-7091. ; 9:3, s. 263-265
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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.
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| 20. |
- Kassemeyer, Stephan, et al.
(författare)
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Femtosecond free-electron laser x-ray diffraction data sets for algorithm development
- 2012
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Ingår i: Optics Express. - 1094-4087. ; 20:4, s. 4149-4158
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Tidskriftsartikel (refereegranskat)abstract
- We describe femtosecond X-ray diffraction data sets of viruses and nanoparticles collected at the Linac Coherent Light Source. The data establish the first large benchmark data sets for coherent diffraction methods freely available to the public, to bolster the development of algorithms that are essential for developing this novel approach as a useful imaging technique. Applications are 2D reconstructions, orientation classification and finally 3D imaging by assembling 2D patterns into a 3D diffraction volume.
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| 21. |
- Kern, Jan, et al.
(författare)
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Room temperature femtosecond X-ray diffraction of photosystem II microcrystals
- 2012
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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
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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.
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| 22. |
- Kern, Jan, et al.
(författare)
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Simultaneous Femtosecond X-ray Spectroscopy and Diffraction of Photosystem II at Room Temperature
- 2013
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 340:6131, s. 491-495
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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.
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| 23. |
- Kern, Jan, et al.
(författare)
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Structures of the intermediates of Kok’s photosynthetic water oxidation clock
- 2018
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 563, s. 421-425
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Tidskriftsartikel (refereegranskat)abstract
- Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3–7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.
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| 24. |
- Kern, Jan, et al.
(författare)
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Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy
- 2014
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5, s. 4371-
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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.
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| 25. |
- Koopmann, Rudolf, et al.
(författare)
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In vivo protein crystallization opens new routes in structural biology
- 2012
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 9:3, s. 259-262
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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.
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| 26. |
- Kubin, Markus, et al.
(författare)
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Soft x-ray absorption spectroscopy of metalloproteins and high-valent metal-complexes at room temperature using free-electron lasers
- 2017
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Ingår i: Structural Dynamics. - : AMER INST PHYSICS. - 2329-7778. ; 4:5
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Tidskriftsartikel (refereegranskat)abstract
- X-ray absorption spectroscopy at the L-edge of 3d transition metals provides unique information on the local metal charge and spin states by directly probing 3d-derived molecular orbitals through 2p-3d transitions. However, this soft x-ray technique has been rarely used at synchrotron facilities for mechanistic studies of metalloenzymes due to the difficulties of x-ray-induced sample damage and strong background signals from light elements that can dominate the low metal signal. Here, we combine femtosecond soft x-ray pulses from a free-electron laser with a novel x-ray fluorescence-yield spectrometer to overcome these difficulties. We present L-edge absorption spectra of inorganic high-valent Mn complexes (Mn similar to 6-15 mmol/l) with no visible effects of radiation damage. We also present the first L-edge absorption spectra of the oxygen evolving complex (Mn4CaO5) in Photosystem II (Mn < 1 mmol/l) at room temperature, measured under similar conditions. Our approach opens new ways to study metalloenzymes under functional conditions. (C) 2017 Author(s).
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| 27. |
- Kupitz, Christopher, et al.
(författare)
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Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser
- 2014
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 513:7517, s. 261-265
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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.
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| 28. |
- Kurta, Ruslan P., et al.
(författare)
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Correlations in Scattered X-Ray Laser Pulses Reveal Nanoscale Structural Features of Viruses
- 2017
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Ingår i: Physical Review Letters. - : American Physical Society. - 0031-9007 .- 1079-7114. ; 119:15
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Tidskriftsartikel (refereegranskat)abstract
- We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates from the expected perfect icosahedral symmetry. Our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.
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| 29. |
- Loh, N. Duane, et al.
(författare)
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Sensing the wavefront of x-ray free-electron lasers using aerosol spheres
- 2013
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Ingår i: Optics Express. - 1094-4087. ; 21:10, s. 12385-12394
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Tidskriftsartikel (refereegranskat)abstract
- Characterizing intense, focused x-ray free electron laser (FEL) pulses is crucial for their use in diffractive imaging. We describe how the distribution of average phase tilts and intensities on hard x-ray pulses with peak intensities of 1021 W/m(2) can be retrieved from an ensemble of diffraction patterns produced by 70 nm-radius polystyrene spheres, in a manner that mimics wavefront sensors. Besides showing that an adaptive geometric correction may be necessary for diffraction data from randomly injected sample sources, our paper demonstrates the possibility of collecting statistics on structured pulses using only the diffraction patterns they generate and highlights the imperative to study its impact on single-particle diffractive imaging.
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| 30. |
- Mitzner, Rolf, et al.
(författare)
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L-Edge X-ray Absorption Spectroscopy of Dilute Systems Relevant to Metalloproteins Using an X-ray Free-Electron Laser
- 2013
-
Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 4:21, s. 3641-3647
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Tidskriftsartikel (refereegranskat)abstract
- L-edge spectroscopy of 3d transition metals provides important electronic structure information and has been used in many fields. However, the use of this method for studying dilute aqueous systems, such as metalloenzymes, has not been prevalent because of severe radiation damage and the lack of suitable detection systems. Here we present spectra from a dilute Mn aqueous solution using a high-transmission zone-plate spectrometer at the Linac Coherent Light Source (LCLS). The spectrometer has been optimized for discriminating the Mn L-edge signal from the overwhelming 0 K-edge background that arises from water and protein itself, and the ultrashort LCLS X-ray pulses can outrun X-ray induced damage. We show that the deviations of the partial-fluorescence yield-detected spectra from the true absorption can be well modeled using the state-dependence of the fluorescence yield, and discuss implications for the application of our concept to biological samples.
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| 31. |
- Park, Hyung Joo, et al.
(författare)
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Toward unsupervised single-shot diffractive imaging of heterogeneous particles using X-ray free-electron lasers
- 2013
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Ingår i: Optics Express. - 1094-4087. ; 21:23, s. 28729-28742
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Tidskriftsartikel (refereegranskat)abstract
- Single shot diffraction imaging experiments via X-ray free-electron lasers can generate as many as hundreds of thousands of diffraction patterns of scattering objects. Recovering the real space contrast of a scattering object from these patterns currently requires a reconstruction process with user guidance in a number of steps, introducing severe bottlenecks in data processing. We present a series of measures that replace user guidance with algorithms that reconstruct contrasts in an unsupervised fashion. We demonstrate the feasibility of automating the reconstruction process by generating hundreds of contrasts obtained from soot particle diffraction experiments.
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| 32. |
- Seibert, M. Marvin, et al.
(författare)
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Single mimivirus particles intercepted and imaged with an X-ray laser
- 2011
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 470:7332, s. 78-81
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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.
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| 33. |
- Sellberg, Jonas A., et al.
(författare)
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How Cubic Can Ice Be?
- 2017
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Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 8:14, s. 3216-3222
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Tidskriftsartikel (refereegranskat)abstract
- Using an X-ray laser, we investigated the crystal structure of ice formed by homogeneous ice nucleation in deeply supercooled water nanodrops (r approximate to 10 nm) at similar to 225 K The nanodrops were formed by condensation of vapor in a supersonic nozzle, and the ice was probed within 100 mu s of freezing using femtosecond wide-angle X-ray scattering at the Linac Coherent Light Source free-electron X-ray laser. The X-ray diffraction spectra indicate that this ice has a metastable, predominantly cubic structure; the shape of the first ice diffraction peak suggests stacking-disordered ice with a cubicity value, chi, in the range of 0.78 +/- 0.05. The cubicity value determined here is higher than those determined in experiments with micron-sized drops but comparable to those found in molecular dynamics simulations. The high cubicity is most likely caused by the extremely low freezing temperatures and by the rapid freezing, which occurs on a similar to 1 mu s time scale in single nanodroplets.
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| 34. |
- Sierra, Raymond G., et al.
(författare)
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Nanoflow electrospinning serial femtosecond crystallography
- 2012
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Ingår i: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 68, s. 1584-1587
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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.
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| 35. |
- Yoon, Chun Hong, et al.
(författare)
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Unsupervised classification of single-particle X-ray diffraction snapshots by spectral clustering
- 2011
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Ingår i: Optics Express. - 1094-4087. ; 19:17, s. 16542-16549
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Tidskriftsartikel (refereegranskat)abstract
- Single-particle experiments using X-ray Free Electron Lasers produce more than 10(5) snapshots per hour, consisting of an admixture of blank shots (no particle intercepted), and exposures of one or more particles. Experimental data sets also often contain unintentional contamination with different species. We present an unsupervised method able to sort experimental snapshots without recourse to templates, specific noise models, or user-directed learning. The results show 90% agreement with manual classification.
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