| 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. |
- 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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| 3. |
- 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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| 4. |
- Bairy, Sneha, et al.
(författare)
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Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies.
- 2018
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Ingår i: Microbial biotechnology. - : Wiley. - 1751-7915. ; 11:2, s. 420-428
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Tidskriftsartikel (refereegranskat)abstract
- The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and invivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization invivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.
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| 5. |
- Caing Carlsson, Rhawnie, et al.
(författare)
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Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum
- 2017
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Ingår i: Acta crystallographica. Section F, Structural biology communications. - 2053-230X. ; 73:6, s. 356-362
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Tidskriftsartikel (refereegranskat)abstract
- Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5′-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.
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| 6. |
- Cellini, Andrea, 1991, et al.
(författare)
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The three-dimensional structure of Drosophila melanogaster (6-4) photolyase at room temperature
- 2021
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Ingår i: Acta Crystallographica Section D-Structural Biology. - : International Union of Crystallography (IUCr). - 2059-7983. ; 77, s. 1001-1009
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Tidskriftsartikel (refereegranskat)abstract
- (6-4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6-4) photolyase [Dm(6-4)photolyase] at room and cryogenic temperatures are reported. The room-temperature structure was solved to 2.27 angstrom resolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79 angstrom resolution using conventional X-ray crystallography. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV-Vis absorption spectroscopy confirms that Dm(6-4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.
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| 7. |
- 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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| 8. |
- Ghosh, Swagatha, 1987, et al.
(författare)
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Modulation of biliverdin dynamics and spectral properties by Sandercyanin
- 2022
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 12:31, s. 20296-20304
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Tidskriftsartikel (refereegranskat)abstract
- Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (Stizostedion vitreum). In this study, we present the structures and binding modes of BV to SFP. Using a combination of designed site-directed mutations, X-ray crystallography, UV/VIS, and resonance Raman spectroscopy, we have identified multiple conformations of BV that are stabilized in the binding pocket of SFP. In complex with the protein, these conformers generate varied spectroscopic signatures both in their absorption and fluorescence spectra. We show that despite no covalent anchor, structural heterogeneity of the chromophore is primarily driven by the D-ring pyrrole of BV. Our work shows how conformational promiscuity of BV is correlated to the rearrangement of amino acids in the protein matrix leading to modulation of spectral properties.
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| 9. |
- Shilova, Anastasiia, et al.
(författare)
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Current status and future opportunities for serial crystallography at MAX IV Laboratory
- 2020
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Ingår i: Journal of Synchrotron Radiation. - Chichester : Wiley-Blackwell. - 0909-0495 .- 1600-5775. ; 27, s. 1095-1102
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Tidskriftsartikel (refereegranskat)abstract
- Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.
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| 10. |
- Yadav, K., et al.
(författare)
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Phenylalanine stacking enhances the red fluorescence of biliverdin IX alpha on UV excitation in sandercyanin fluorescent protein
- 2022
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Ingår i: Febs Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 596:6, s. 796-805
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Tidskriftsartikel (refereegranskat)abstract
- Biliverdin IX alpha (BV) binds to several prokaryotic and eukaryotic proteins. How nature exploits the versatility of BV's properties is not fully understood. Unlike free BV, the Sandercyanin fluorescent protein bound to BV (SFP-BV) shows enhanced red fluorescence (675 nm) on excitation in the UV region (380 nm). Site-directed mutagenesis showed that the BV complex of two SFP variants, F55A and E79A, resulted in the loss of red fluorescence. Crystal structures of the complexes of these proteins with BV show the absence of stacking interactions of the F55 phenyl ring with BV. BV changes from ZZZssa conformation in the wild-type to ZZZsss conformation in the variants. In the nonfluorescent mutants, the lowest excited state is destabilized, resulting in nonradiative decay.
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