| 1. |
- Båth, Petra, 1988, et al.
(författare)
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Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre
- 2022
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Ingår i: Acta Crystallographica Section D-Structural Biology. - : International Union of Crystallography (IUCr). - 2059-7983. ; 78, s. 698-708
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Tidskriftsartikel (refereegranskat)abstract
- Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 angstrom resolution when exposed to XFEL radiation, which is an improvement of 0.15 angstrom over previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile Q(B) pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.
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| 2. |
- 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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| 3. |
- 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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| 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. |
- Johansson, Linda C, 1983, et al.
(författare)
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Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography.
- 2013
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4
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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.
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| 6. |
- Malmerberg, Erik, 1980, et al.
(författare)
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Conformational activation of visual rhodopsin in native disc membranes
- 2015
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 8:367
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Tidskriftsartikel (refereegranskat)abstract
- Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested.
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| 7. |
- Nango, E., et al.
(författare)
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A three-dimensional movie of structural changes in bacteriorhodopsin
- 2016
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 354:6319, s. 1552-1557
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.
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| 8. |
- Wickstrand, Cecilia, et al.
(författare)
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A tool for visualizing protein motions in time-resolved crystallography
- 2020
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Ingår i: Structural Dynamics. - : AIP Publishing. - 2329-7778. ; 7:2
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Tidskriftsartikel (refereegranskat)abstract
- Time-resolved serial femtosecond crystallography (TR-SFX) at an x-ray free electron laser enables protein structural changes to be imaged on time-scales from femtoseconds to seconds. It can, however, be difficult to grasp the nature and timescale of global protein motions when structural changes are not isolated near a single active site. New tools are, therefore, needed to represent the global nature of electron density changes and their correlation with modeled protein structural changes. Here, we use TR-SFX data from bacteriorhodopsin to develop and validate a method for quantifying time-dependent electron density changes and correlating them throughout the protein. We define a spherical volume of difference electron density about selected atoms, average separately the positive and negative electron difference densities within each volume, and walk this spherical volume through all atoms within the protein. By correlating the resulting difference electron density amplitudes with time, our approach facilitates an initial assessment of the number and timescale of structural intermediates and highlights quake-like motions on the sub-picosecond timescale. This tool also allows structural models to be compared with experimental data using theoretical difference electron density changes calculated from refined resting and photo-activated structures.
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| 9. |
- Wickstrand, Cecilia, et al.
(författare)
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Bacteriorhodopsin: Structural insights revealed using x-ray lasers and synchrotron radiation
- 2019
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Ingår i: Annual Review of Biochemistry. - : Annual Reviews. - 0066-4154 .- 1545-4509. ; 88, s. 59-83
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Forskningsöversikt (refereegranskat)abstract
- Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.
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| 10. |
- Wickstrand, Cecilia, et al.
(författare)
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Bacteriorhodopsin: Would the real structural intermediates please stand up?
- 2015
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Ingår i: Biochimica Et Biophysica Acta-General Subjects. - : Elsevier BV. - 0304-4165. ; 1850:3, s. 536-553
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Tidskriftsartikel (refereegranskat)abstract
- Background: Bacteriorhodopsin (bR) is the simplest known light driven proton pump and has been heavily studied using structural methods: eighty four X-ray diffraction, six electron diffraction and three NMR structures of bR are deposited within the protein data bank. Twenty one X-ray structures report light induced structural changes and changes induced by mutation, changes in pH, thermal annealing or X-ray induced photoreduction have also been examined. Scope of review: We argue that light-induced structural changes that are replicated across several studies by independent research groups are those most likely to represent what is happening in reality. We present both internal distance matrix analyses that sort deposited bR structures into hierarchal trees, and difference Fourier analysis of deposited X-ray diffraction data. Major conclusions: An internal distance matrix analysis separates most wild-type bR structures according to their different crystal forms, indicating how the protein's structure is influenced by crystallization conditions. A similar analysis clusters eleven studies of illuminated bR crystals as one branch of a hierarchal tree with reproducible movements of the extracellular portion of helix C towards helix G, and of the cytoplasmic portion of helix F away from helices A, B and G. All crystallographic data deposited for illuminated crystals show negative difference density on a water molecule (Wat402) that forms H-bonds to the retinal Schiff Base and two aspartate residues (Asp85, Asp212) in the bR resting state. Other recurring difference density features indicated reproducible side-chain, backbone and water molecule displacements. X-ray induced radiation damage also disorders Wat402 but acts via cleaving the head-groups of Asp85 and Asp212. General significance: A remarkable level of agreement exists when deposited structures and crystallographic observations are viewed as a whole. From this agreement a unified picture of the structural mechanism of light-induced proton pumping by bR emerges. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins. (C) 2014 The Authors. Published by Elsevier B.V.
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| 11. |
- Wickstrand, Cecilia
(författare)
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Production and Structural Dynamics of Microbial Rhodopsins
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rhodopsins are a family of membrane proteins that are found in a wide range of organisms and provide them with the ability to sense and react to light. When light is absorbed by these proteins, structural changes occur that initiate the pumping of protons, opening of a channel or signal transfer. The rhodopsins have different functions but they are structurally similar. This makes the protein family interesting from the perspective of structural biology, a field that aims to link function of proteins to structure and dynamics. The structure of a protein may be obtained with X-ray crystallography. It requires a pure protein sample that can form protein crystals, data collection at an X-ray source and analysis of data for structural modelling and interpretation. In the last ten years, the development of X-ray free electron lasers (XFELs) generating very intense and short X-ray pulses has made it possible to capture structural changes in real-time with a time resolution of femtoseconds. This technique, called time-resolved serial femtosecond crystallography (TR-SFX), is particularly suited for studying structural dynamics of light-activated proteins. The first part of this thesis is about production of channelrhodopsins. Channelrhodopsins are light-gated ion channels that we aim to produce for a future TR-SFX experiment. We describe protein production in the yeast Pichia pastoris and strategies to handle glycosylations of the proteins. We also establish protocols for purification and screen for microcrystals in the lipidic cubic phase. The second and major part of this thesis is about structural dynamics of bacteriorhodopsin, a light-driven proton pump and by far the most studied microbial rhodopsin. We perform a TR-SFX study on bacteriorhodopsin that reveal structural changes in the time window from nanoseconds to milliseconds. In addition, we develop tools for comparison and analysis of structures and difference Fourier electron density maps from intermediate trapping studies and TR-SFX. These analyses give new insights in the mechanism of proton pumping.
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