| 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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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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| 3. |
- Andersson, Rebecka, 1988, et al.
(författare)
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Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature.
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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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.
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| 4. |
- Andersson, Rebecka, 1988, et al.
(författare)
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Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments.
- 2019
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Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 75:Pt 10, s. 937-946
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Tidskriftsartikel (refereegranskat)abstract
- Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.
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| 5. |
- 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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| 6. |
- Edlund, Petra, et al.
(författare)
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The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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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.
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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. |
- 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. |
- Aponte-Santamaria, C., et al.
(författare)
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Temperature dependence of protein-water interactions in a gated yeast aquaporin
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Regulation of aquaporins is a key process of living organisms to counteract sudden osmotic changes. Aqy1, which is a water transporting aquaporin of the yeast Pichia pastoris, is suggested to be gated by chemo-mechanical stimuli as a protective regulatory-response against rapid freezing. Here, we tested the influence of temperature by determining the X-ray structure of Aqy1 at room temperature (RT) at 1.3 angstrom resolution, and by exploring the structural dynamics of Aqy1 during freezing through molecular dynamics simulations. At ambient temperature and in a lipid bilayer, Aqy1 adopts a closed conformation that is globally better described by the RT than by the low-temperature (LT) crystal structure. Locally, for the blocking-residue Tyr31 and the water molecules inside the pore, both LT and RT data sets are consistent with the positions observed in the simulations at room-temperature. Moreover, as the temperature was lowered, Tyr31 adopted a conformation that more effectively blocked the channel, and its motion was accompanied by a temperature-driven rearrangement of the water molecules inside the channel. We therefore speculate that temperature drives Aqy1 from a loosely-to a tightly-blocked state. This analysis provides high-resolution structural evidence of the influence of temperature on membrane-transport channels.
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| 10. |
- Båth, Petra, 1988
(författare)
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Micro-Crystallization and Time-Resolved Diffraction Studies of a Bacterial Photosynthetic Reaction Center
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Photosynthesis is one of the most important set of chemical reactions in nature as they can convert sunlight into hydrocarbons and chemical energy. The proteins responsible for this are two general types of reaction centers that can be found in a wide variety of living organisms capable of photosynthesis, from bacteria to algae and plants. Despite the range of host cells the reaction centers themselves have fairly conserved structure and function where the absorption of light leads to an electron transfer process and eventually the production of energy. The work in this thesis is focused on the bacterial reaction center from Blastochloris viridis, which is an analogue to photosystem II in plants. Our studies aimed to further examine exactly what happens in the protein as light is absorbed. X-ray crystallography has been an important tool for determining the atomic structure of proteins for several decades. This technique requires that the protein in question is in a crystalline form or else no structural data can be obtained. The development of a new generation of X-ray sources, X-ray free-electron lasers, makes new types of experiments possible but it also requires new ways of preparing crystals for the highly specialized delivery systems used. This thesis presents new ways of preparing membrane protein microcrystals for different types of delivery media. A new way to make crystals in lipidic cubic phase is presented based on setting up crystallization trials in deep-well plates and vials rather than the standard gas-tight syringes. This basic protocol has been developed to add crystal seeds as well as making crystals in an oxygen-free environment. Using this method a 2.3 Å resolution X-ray structure of reaction center was obtained from seeded crystals measuring only 20 μm. For crystals growing in vapour diffusion several techniques of generating crystals are presented depending on how far the screening protocols have been developed; initial crystals can simply be crushed into the size required and more homogeneous microcrystals can be produced by a seeding protocol. These crystals were then used in a time resolved study at an XFEL showing the structural movements of the cofactors in the protein picoseconds after photon absorption.
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| 11. |
- 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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| 12. |
- Nogly, P., et al.
(författare)
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Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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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.
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| 13. |
- Nogly, P., et al.
(författare)
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Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser
- 2018
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 361:6398
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION Retinal is a light-sensitive protein ligand that is used by all domains of life to process the information and energy content of light. Retinal-binding proteins are integral membrane proteins that drive vital biological processes, including light sensing for spatial orientation and circadian clock adjustment, as well as maintaining electrochemical gradients through ion transport. They also form the basis for optogenetic manipulation of neural cells. How the protein environment guides retinal isomerization on a subpicosecond time scale toward a single high-yield product is a fundamental outstanding question in photobiology. RATIONALE Light-induced isomerization of retinal is among the fastest reactions known in biology. It has been widely studied by spectroscopic techniques to probe the evolution of spectral intermediates over time. Using x-ray free-electron lasers (XFELs), it is now possible to observe ultrafast photochemical reactions and their induced molecular motions within proteins on scales of femtoseconds to milliseconds with near-atomic structural resolution. In this work, we used XFEL radiation to study the structural dynamics of retinal isomerization in the light-driven proton-pump bacteriorhodopsin (bR). The principal mechanism of isomerization in this prototypical retinal-binding protein has direct relevance for all other members of this important family of membrane proteins, and it provides insight into how protein environments catalyze photochemical reactions in general. RESULTS We collected high-resolution x-ray diffraction data from bR microcrystals injected across the femtosecond x-ray pulses of the Linac Coherent Light Source after excitation of the retinal chromophore by an optical laser pulse. X-ray diffraction images were sorted into temporal subgroups with a precision of about 200 fs. A series of 18 overlapping difference Fourier electron density maps reveal structural changes over the first picosecond of retinal photoexcitation. Complementary data for time delays of 10 ps and 8.33 ms allow us to resolve the later stages of the reaction. In combination with refined crystallographic structures at pump-probe delays corresponding to where the spectroscopically characterized I, J, K, and M intermediates form in solution, our time-resolved structural data reveal the trajectory of retinal isomerization and provide atomic details at key points along the reaction. The aspartic acid residues of the retinal counterion and functional water molecules in close proximity to the retinal Schiff base respond collectively to the formation and decay of the excited state. This collective motion sets the stage for retinal isomerization, which proceeds via a twisted retinal configuration. Quantum mechanics/molecular mechanics simulations provide theoretical support for this structural evolution. CONCLUSION Our observations reveal how, concomitant with the formation of the earliest excited state, the retinal-binding pocket opens up in close proximity to the isomerizing bond. We propose that ultrafast charge transfer along retinal is a driving force for collective motions that contribute to the stereoselectivity and efficiency of retinal isomerization within a protein scaffold. Vibrational quake-like motions extending from retinal to the protein may also be a mechanism through which excess energy is released in a nonradiative fashion.
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| 14. |
- Sjöhamn, Jennie, 1986, et al.
(författare)
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Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes.
- 2016
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Ingår i: Protein science : a publication of the Protein Society. - : Wiley. - 1469-896X. ; 25:12, s. 2196-2208
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Tidskriftsartikel (refereegranskat)abstract
- Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high-resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0-CaM complex on the AQP0 C-terminus is also demonstrated since the C-terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X-ray crystallography or single particle electron microscopy.
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