| 1. |
- Popp, David, et al.
(författare)
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Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser
- 2017
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Ingår i: CYTOSKELETON. - : WILEY. - 1949-3584 .- 1949-3592. ; 74:12, s. 472-481
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Tidskriftsartikel (refereegranskat)abstract
- A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked -strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual -synuclein amyloids.
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| 2. |
- Brändén, Gisela, 1975, et al.
(författare)
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Coherent diffractive imaging of microtubules using an X-ray laser.
- 2019
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.
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| 3. |
- Aquila, Andrew, et al.
(författare)
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Time-resolved protein nanocrystallography using an X-ray free-electron laser
- 2012
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Ingår i: Optics Express. - 1094-4087. ; 20:3, s. 2706-2716
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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.
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| 4. |
- Arnlund, David, et al.
(författare)
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Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser
- 2014
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 11:9, s. 923-926
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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.
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| 5. |
- Arnlund, David
(författare)
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X-ray free-electron laser based methods for structural and ultrafast dynamics studies of a photosynthetic reaction centre
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Life on earth is fuelled by the energy of sunlight, which must first be captured and converted into a chemical energy form useful to the cell. This process is known as photosynthesis and the major pathway of this energy conversion is via photosynthetic reaction centres. These enzymes convert the energy content of an absorbed photon into a transmembrane potential difference via the movements of electrons. Increasing our knowledge of the three-dimensional fold and structural changes that takes place within photosynthetic reaction centres is therefore of considerable importance for understanding biological photosynthesis. The aim of this work has been to adapt methods for both crystallographic and solution phase structural studies of membrane proteins to the unique properties of X-ray free-electron laser (XFEL) radiation. To accomplish this, a new crystallization technique for the photosynthetic reaction centre from the purple bacterium Blastochloris viridis (RCvir) was developed which was suitable for serial femtosecond crystallography (SFX) experiments at an XFEL. Our initial experiments at the Linac Coherent Light Source (LCLS), the world’s first XFEL, yielded an SFX structure of RCvir to 8.2 Å resolution. After the LCLS decreased the X-ray wavelength at which the facility could operate, and in combination with improved crystallization conditions, we later resolved the SFX structure of RCvir to 3.5 Å resolution. Whether or not ultrafast structural changes in RCvir occur in photosynthesis has been debated for two decades. We addressed this question by developing time-resolved wide-angle X-ray scattering (TR-WAXS) studies at the LCLS that could capture rapid structural changes in solubilized samples of RCvir. Proof-of-principle experiments revealed a structural deformation that propagated through the RCvir protein following multi-photon absorption by its cofactors, enabling a protein quake through a photosynthetic protein to be visualized. Further insight was provided by a second TR-WAXS experiment in which this structural signal was observed in the data as the pump laser fluence was decreased to less than one photon absorbed per RCvir molecule. This result implies that, even under physiological conditions of normal sunlight, ultrafast protein structural rearrangements may influence the primary charge separation events of biological photosynthesis.
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| 6. |
- Barty, A., et al.
(författare)
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Self-terminating diffraction gates femtosecond X-ray nanocrystallography measurements
- 2012
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Ingår i: Nature Photonics. - 1749-4885 .- 1749-4893. ; 6:1, s. 35-40
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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.
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| 7. |
- Boutet, S., et al.
(författare)
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High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography
- 2012
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 337:6092, s. 362-364
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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.
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| 8. |
- Dods, Robert, 1989, et al.
(författare)
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From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.
- 2017
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Ingår i: Structure. - : Elsevier BV. - 1878-4186 .- 0969-2126. ; 25:9, s. 1461-1468
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Tidskriftsartikel (refereegranskat)abstract
- Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.
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| 9. |
- Dods, Robert, 1989, et al.
(författare)
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Ultrafast structural changes within a photosynthetic reaction centre.
- 2021
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 589:7841, s. 310-314
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Tidskriftsartikel (refereegranskat)abstract
- Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.
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| 10. |
- 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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