| 1. |
- Brändén, Gisela, 1975, et al.
(författare)
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Advances and challenges in time-resolved macromolecular crystallography.
- 2021
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 373:6558
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Forskningsöversikt (refereegranskat)abstract
- Conformational changes within biological macromolecules control a vast array of chemical reactions in living cells. Time-resolved crystallography can reveal time-dependent structural changes that occur within protein crystals, yielding chemical insights in unparalleled detail. Serial crystallography approaches developed at x-ray free-electron lasers are now routinely used for time-resolved diffraction studies of macromolecules. These techniques are increasingly being applied at synchrotron radiation sources and to a growing diversity of macromolecules. Here, we review recent progress in the field, including visualizing ultrafast structural changes that guide the initial trajectories of light-driven reactions as well as capturing biologically important conformational changes on slower time scales, for which bacteriorhodopsin and photosystem II are presented as illustrative case studies.
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| 2. |
- 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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| 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. |
- Ghosh, Swagatha, 1987, et al.
(författare)
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A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography
- 2023
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Ingår i: Journal of Applied Crystallography. - : International Union of Crystallography (IUCr). - 1600-5767. ; 56, s. 449-460
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Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography was initially developed for roomtemperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometercompatible 3D-printed support and is connected to a syringe pump via lightweight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 angstrom resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.
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| 5. |
- Ghosh, Swagatha, et al.
(författare)
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A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography
- 2023
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Ingår i: Journal of Applied Crystallography. - 0021-8898. ; 56:Pt 2, s. 449-460
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Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via lightweight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 Å resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.
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| 6. |
- Gruhl, T., et al.
(författare)
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Ultrafast structural changes direct the first molecular events of vision
- 2023
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 615, s. 939-944
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Tidskriftsartikel (refereegranskat)abstract
- Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)(1). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation(2), thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature(3) to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.
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| 7. |
- Panagaki, Dimitra, et al.
(författare)
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Nuclear envelope budding is a response to cellular stress.
- 2021
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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. - 1091-6490 .- 0027-8424. ; 118:30
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Tidskriftsartikel (refereegranskat)abstract
- Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.
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| 8. |
- Safari, Cecilia, 1989, et al.
(författare)
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Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase
- 2023
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Ingår i: Science advances. - 2375-2548. ; 9:49
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.
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| 9. |
- Sandelin, Emil, 1994, et al.
(författare)
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Characterization and evaluation of photolabile (μ-peroxo)(μ-hydroxo)bis[bis(bipyridyl)cobalt caged oxygen compounds to facilitate time-resolved crystallographic studies of cytochrome c oxidase
- 2024
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Ingår i: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES. - 1474-905X .- 1474-9092. ; 23, s. 839-851
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Tidskriftsartikel (refereegranskat)abstract
- Photolabile (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)-cobalt-based caged oxygen compounds have been synthesized and characterized by optical absorbance spectroscopy, X-ray crystallography. and the quantum yield and redox stability were investigated. Furthermore, conditions were established where redox incompatibilities encountered between caged oxygen compounds and oxygen-dependant cytochrome c oxidase (CcO) could be circumvented. Herein, we demonstrate that millimolar concentrations of molecular oxygen can be released from a caged oxygen compound with spatio-temporal control upon laser excitation, triggering enzymatic turnover in cytochrome c oxidase. Spectroscopic evidence confirms the attainment of a homogeneous reaction initiation at concentrations and conditions relevant for further crystallography studies. This was demonstrated by the oxidizing microcrystals of reduced CcO by liberation of millimolar concentrations of molecular oxygen from a caged oxygen compound. We believe this will expand the scope of available techniques for the detailed investigation of oxygen-dependant enzymes with its native substrate and facilitate further time-resolved X-ray based studies such as wide/small angle X-ray scattering and serial femtosecond crystallography.
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| 10. |
- Sandelin, Emil, et al.
(författare)
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Spatiotemporal Release of Singlet Oxygen in Low Molecular Weight Organo-Gels Upon Thermal or Photochemical External Stimuli
- 2024
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Ingår i: Small. - 1613-6810.
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Tidskriftsartikel (refereegranskat)abstract
- The first example of a material capable of spatiotemporal catch and release of singlet oxygen (1O2) in gel phase is presented. Several low molecular weight organogelators based around an oxotriphenylhexanoate (OTHO) core are developed and optimized with regard to; their gelation properties, and ability of releasing 1O2 upon thermal and/or photochemical external stimuli, in both gel phase and solution. Remarkably, reversible phase transitioning between the gel and solution phase are also demonstrated. Taken together two complementary modes of releasing 1O2, one thermally controlled over time, and one rapid release by means of photochemical stimuli is disclosed. These findings represent the first phase reversible system where function and aggregation properties can be controlled independently, and thus pave the way for novel applications in material sciences as well as in life sciences.
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