| 1. |
- Båth, Petra, 1988, et al.
(författare)
-
Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre
- 2022
-
Ingår i: Acta Crystallographica Section D-Structural Biology. - : International Union of Crystallography (IUCr). - 2059-7983. ; 78, s. 698-708
-
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.
|
|
| 2. |
- Dods, Robert, 1989, et al.
(författare)
-
From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.
- 2017
-
Ingår i: Structure. - : Elsevier BV. - 1878-4186 .- 0969-2126. ; 25:9, s. 1461-1468
-
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.
|
|
| 3. |
- Dods, Robert, 1989, et al.
(författare)
-
Ultrafast structural changes within a photosynthetic reaction centre.
- 2021
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 589:7841, s. 310-314
-
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.
|
|
| 4. |
- Andersson, Rebecka, 1988, et al.
(författare)
-
Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature.
- 2017
-
Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
-
Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba 3-type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba 3-type and aa 3-type cytochrome c oxidases around the proton-loading site are also described.
|
|
| 5. |
- Edlund, Petra, et al.
(författare)
-
The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography
- 2016
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 angstrom resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 angstrom resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.
|
|
| 6. |
- Dods, Robert, 1989, et al.
(författare)
-
CHAPTER 6: Elucidating Ultrafast Structural Motions in Photosynthetic Reaction Centers with XFEL Radiation
- 2017
-
Ingår i: RSC Energy and Environment Series. - Cambridge : Royal Society of Chemistry. - 2044-0774. ; , s. 128-140
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Photosynthetic reaction centers are integral membrane proteins found in photosynthetic plants and bacteria and are the work-horses of photosynthesis. Within these proteins, the energy of sunlight is directed to a special pair of closely spaced chlorophyll molecules that become photo-oxidized as an electron is dispelled to the opposite side of the biological membrane. Evolution has optimized the charge separation reactions of photosynthetic reaction centers so as to achieve a remarkably high quantum yield and energy efficiency. Whether or not ultrafast structural changes play any functional role in guiding the primary charge separation reactions of photosynthesis has been debated for two decades but is not yet resolved. Here, we review progress towards visualizing structural changes in photosynthetic reaction centers using synchrotron radiation. We further describe the revolutionary potential of X-ray free electron lasers (XFELs) for shedding new light on ultrafast structural changes in biomolecules. We emphasize milestones towards the goal of observing functionally important ultrafast motions in photosynthetic reaction centers in real time and anticipate that novel high-resolution structural insights are now technically within reach. © The Royal Society of Chemistry 2017.
|
|
| 7. |
- Dods, Robert, 1989
(författare)
-
Ultrafast Structural Changes in a Bacterial Photosynthetic Reaction Center probed with XFEL Radiation
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Photosynthesis is the process by which plants and many species of bacteria convert energy from sunlight into chemical energy used to power their metabolism. As these plants and bacteria are eaten, the chemical energy moves up the food chain and thus photosynthesis provides fuel for almost all life on Earth. Photosynthetic reaction centers are the workhorses of photosynthesis. Upon photo-excitation, these multi-domain integral membrane proteins drive an electron transport chain that results in a proton gradient across the cell membrane. The primary electron transport events are of great interest to the scientific community due to their near perfect efficiency and functional role in powering the biosphere. The articles that comprise this thesis deal with one such photosynthetic reaction center, that from the purple non-sulfur bacterium Blastochloris viridis (RCvir). Spectroscopic studies of RCvir have revealed that the initial charge-separation reactions occur on a time scale of picoseconds and raise interesting questions about the role of ultrafast structural changes in optimizing the efficiency of the overall process. As X-ray free-electron lasers (XFELs) have been commissioned, the possibility of studying the initial light-driven reactions of the electron transport process through time-resolved crystallography has been realized. XFELs are powerful new X-ray sources that have a high peak brilliance and a pulse length three orders of magnitude shorter than the most advanced synchrotron source. Through the development of time-resolved crystallographic and solution scattering methods at XFELs, this thesis aims to deliver new information about the role structural changes play in guiding the charge separation reactions of photosynthesis. A solution scattering experiment was performed to give physiological relevance to previous observations that multi-photon excitation led to quake like movements within RCvir on the order of picoseconds. Oscillatory features were revealed following a single-photon absorption event, but these proved difficult to interpret structurally. This highlighted the need for time-resolved crystallography experiments that could directly visualize these structural changes. After optimizing crystallization methods to produce samples suitable for XFEL sources, a time-resolved crystallography experiment was conducted that captured the protein at two picosecond time-points following photo-excitation. These experiments allowed visualization of conformational changes that evolved over time and it is hypothesized these structural dynamics may play a role in altering the activation energies of the electron transport process.
|
|
| 8. |
- Nango, E., et al.
(författare)
-
A three-dimensional movie of structural changes in bacteriorhodopsin
- 2016
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 354:6319, s. 1552-1557
-
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.
|
|
| 9. |
- Nogly, P., et al.
(författare)
-
Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography
- 2016
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 angstrom resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
|
|
| 10. |
- Wickstrand, Cecilia, et al.
(författare)
-
Bacteriorhodopsin: Would the real structural intermediates please stand up?
- 2015
-
Ingår i: Biochimica Et Biophysica Acta-General Subjects. - : Elsevier BV. - 0304-4165. ; 1850:3, s. 536-553
-
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.
|
|
