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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. |
- 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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| 6. |
- 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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| 7. |
- Brändén, Gisela, 1975-
(författare)
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Structure and Function of Cytochrome c Oxidase
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aerobic organisms, ranging from bacteria to humans, obtain their energy through the process of respiration. Electrons from the breakdown of food are transported through the membrane-bound enzyme complexes of the respiratory chain, to the terminal acceptor molecule, oxygen. The energy released in the process is used to transport protons across the cell membrane, thereby establishing an electrochemical gradient that can be utilised e.g. to synthesise ATP. The last respiratory complex in mitochondria and many bacteria is cytochrome c oxidase (CcO). CcO catalyses the reduction of oxygen to water, and at the same time pumps protons from the more negative to the more positive side of the membrane. In spite of extended and intensive research efforts, the detailed mechanism by which the exergonic electron-transfer reactions are coupled to the endergonic proton pumping is still not understood.The focus of this thesis is the structure and function of CcO from the bacterium Rhodobacter sphaeroides. The structures of the wild-type enzyme and two mutant enzymes have been investigated, providing insights into structural changes taking place upon replacing a glutamate residue (E286) in one of the proton-transfer pathways. E286 is a key element in directing protons either to the catalytic site for the oxygen chemistry or towards the outside for pumping. Results from time-resolved optical absorption spectroscopy show that the pKa of E286 has to be close to 9.5 for the proton pump to function, and that the pKa can be modulated by mutations at a distance of ~25 Å from E286. From another study we conclude that the D-propionate of haem a3 is most likely the acceptor site for pumped protons. Finally, we have identified the structural element which conformational changes determine the reaction rate of each proton-pumping step.The results are summarised in a molecular model for proton pumping by CcO, in which deprotonation of E286 drives proton translocation across the membrane.
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| 8. |
- Brändén, Gisela, 1975, et al.
(författare)
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Structure-based ligand design to overcome CYP inhibition in drug discovery projects
- 2014
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Ingår i: Drug Discovery Today. - : Elsevier BV. - 1359-6446. ; 19:7, s. 905-911
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Forskningsöversikt (refereegranskat)abstract
- Cytochrome P450 (CYP) enzymes are key players in xenobiotic metabolism, and inhibition of CYPs can therefore result in unwanted drug-drug interactions. Within drug discovery, CYP inhibition can cause delays in the progression of candidate drugs, or even premature closure of projects. During the past decade, a massive effort in the pharmaceutical industry and academic research has produced a wealth of structural information in the CYP field. In this short review, we will describe how structure-based approaches can be used in the pharmaceutical industry to work away from CYP inhibition, with a focus on the opportunities and challenges. We will show two examples from our own work where structural information on CYP2C9 and CYP3A4 inhibitor complexes have been successfully exploited in ongoing drug discovery projects.
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| 9. |
- 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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| 10. |
- 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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