| 1. |
- Agirre, Jon, et al.
(författare)
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The CCP4 suite: integrative software for macromolecular crystallography
- 2023
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Ingår i: Acta Crystallographica Section D. - : INT UNION CRYSTALLOGRAPHY. - 2059-7983. ; 79, s. 449-461
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Tidskriftsartikel (refereegranskat)abstract
- The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.
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| 2. |
- Ahlqvist, Josefin, et al.
(författare)
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Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6
- 2022
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Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 78:Pt 2, s. 212-227
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Tidskriftsartikel (refereegranskat)abstract
- This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.
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| 3. |
- Ahlqvist, Josefin, et al.
(författare)
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Crystal structure of DNA polymerase I from Thermus phage G20c
- 2022
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Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 78:Pt 11, s. 1384-1398
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Tidskriftsartikel (refereegranskat)abstract
- This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.
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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. |
- Bjelčić, Monika, et al.
(författare)
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Anaerobic fixed-target serial crystallography using sandwiched silicon nitride membranes
- 2023
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 79:Pt 11, s. 1018-1025
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, the emergence of serial crystallography, initially pioneered at X-ray free-electron lasers (XFELs), has sparked a growing interest in collecting macromolecular crystallographic data at room temperature. Various fixed-target serial crystallography techniques have been developed, ranging from commercially available chips to in-house designs implemented at different synchrotron facilities. Nevertheless, there is currently no commercially available chip (known to the authors) specifically designed for the direct handling of oxygen-sensitive samples. This study presents a methodology employing silicon nitride chips arranged in a 'sandwich' configuration, enabling reliable room-temperature data collection from oxygen-sensitive samples. The method involves the utilization of a custom-made 3D-printed assembling tool and a MX sample holder. To validate the effectiveness of the proposed method, deoxyhemoglobin and methemoglobin samples were investigated using the BioMAX X-ray macromolecular crystallography beamline, the Balder X-ray absorption spectroscopy beamline and UV-Vis absorption spectroscopy.
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| 6. |
- Buts, Lieven, et al.
(författare)
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Impact of natural variation in bacterial F17G adhesins on crystallization behaviour.
- 2005
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Ingår i: Acta crystallographica. Section D, Biological crystallography. - : International Union of Crystallography (IUCr). - 0907-4449. ; 61:8, s. 1149-1159
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Tidskriftsartikel (refereegranskat)abstract
- Since the introduction of structural genomics, the protein has been recognized as the most important variable in crystallization. Recent strategies to modify a protein to improve crystal quality have included rationally engineered point mutations, truncations, deletions and fusions. Five naturally occurring variants, differing in 1-18 amino acids, of the 177-residue lectin domain of the F17G fimbrial adhesin were expressed and purified in identical ways. For four out of the five variants crystals were obtained, mostly in non-isomorphous space groups, with diffraction limits ranging between 2.4 and 1.1 A resolution. A comparative analysis of the crystal-packing contacts revealed that the variable amino acids are often involved in lattice contacts and a single amino-acid substitution can suffice to radically change crystal packing. A statistical approach proved reliable to estimate the compatibilities of the variant sequences with the observed crystal forms. In conclusion, natural variation, universally present within prokaryotic species, is a valuable genetic resource that can be favourably employed to enhance the crystallization success rate with considerably less effort than other strategies.
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| 7. |
- Buts, L, et al.
(författare)
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Solving the phase problem for carbohydrate-binding proteins using selenium derivatives of their ligands : a case study involving the bacterial F17-G adhesin
- 2003
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Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography (IUCr). - 2059-7983. ; 59:6, s. 1012-1015
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Tidskriftsartikel (refereegranskat)abstract
- The Escherichia coli adhesin F17-G is a carbohydrate-binding protein that allows the bacterium to attach to the intestinal epithelium of young ruminants. The structure of the 17 kDa lectin domain of F17-G was determined using the anomalous dispersion signal of a selenium-containing analogue of the monosaccharide ligand N-acetyl-D-glucosamine in which the anomeric oxygen was replaced by an Se atom. A three-wavelength MAD data set yielded good experimental phases to 2.6 Angstrom resolution. The structure was refined to 1.75 Angstrom resolution and was used to solve the structures of the ligand-free protein and the F17-G-N-acetyl-D-glucosamine complex. This selenium-carbohydrate phasing method could be of general use for determining the structures of carbohydrate-binding proteins.
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| 8. |
- 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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| 9. |
- Caldararu, Octav, et al.
(författare)
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Exploring ligand dynamics in protein crystal structures with ensemble refinement
- 2021
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 1099-1115
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein-ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein-ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and R free values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.
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| 10. |
- Caldararu, Octav, et al.
(författare)
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Refinement of protein structures using a combination of quantum-mechanical calculations with neutron and X-ray crystallographic data
- 2019
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 75, s. 368-380
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Tidskriftsartikel (refereegranskat)abstract
- Neutron crystallography is a powerful method to determine the positions of H atoms in macromolecular structures. However, it is sometimes hard to judge what would constitute a chemically reasonable model, and the geometry of H atoms depends more on the surroundings (for example the formation of hydrogen bonds) than heavy atoms, so that the empirical geometry information for the H atoms used to supplement the experimental data is often less accurate. These problems may be reduced by using quantum-mechanical calculations. A method has therefore been developed to combine quantum-mechanical calculations with joint crystallographic refinement against X-ray and neutron data. A first validation of this method is provided by re-refining the structure of the galectin-3 carbohydrate-recognition domain in complex with lactose. The geometry is improved, in particular for water molecules, for which the method leads to better-resolved hydrogen-bonding interactions. The method has also been applied to the active copper site of lytic polysaccharide monooxygenase and shows that the protonation state of the amino-terminal histidine residue can be determined.
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| 11. |
- Cao, Lili, et al.
(författare)
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Quantum refinement with multiple conformations : Application to the P-cluster in nitrogenase
- 2020
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 76, s. 1145-1156
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Tidskriftsartikel (refereegranskat)abstract
- X-ray crystallography is the main source of atomistic information on the structure of proteins. Normal crystal structures are obtained as a compromise between the X-ray scattering data and a set of empirical restraints that ensure chemically reasonable bond lengths and angles. However, such restraints are not always available or accurate for nonstandard parts of the structure, for example substrates, inhibitors and metal sites. The method of quantum refinement, in which these empirical restraints are replaced by quantum-mechanical (QM) calculations, has previously been suggested for small but interesting parts of the protein. Here, this approach is extended to allow for multiple conformations in the QM region by performing separate QM calculations for each conformation. This approach is shown to work properly and leads to improved structures in terms of electron-density maps and real-space difference density Z-scores. It is also shown that the quality of the structures can be gauged using QM strain energies. The approach, called ComQumX-2QM, is applied to the P-cluster in two different crystal structures of the enzyme nitrogenase, i.e. an Fe 8 S 7 Cys 6 cluster, used for electron transfer. One structure is at a very high resolution (1.0 Å) and shows a mixture of two different oxidation states, the fully reduced P N state (Fe 8 2+, 20%) and the doubly oxidized P 2+ state (80%). In the original crystal structure the coordinates differed for only two iron ions, but here it is shown that the two states also show differences in other atoms of up to 0.7 Å. The second structure is at a more modest resolution, 2.1 Å, and was originally suggested to show only the one-electron oxidized state, P 1+. Here, it is shown that it is rather a 50/50% mixture of the P 1+ and P 2+ states and that many of the Fe - Fe and Fe - S distances in the original structure were quite inaccurate (by up to 0.8 Å). This shows that the new ComQumX-2QM approach can be used to sort out what is actually seen in crystal structures with dual conformations and to give locally improved coordinates.
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| 12. |
- 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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| 13. |
- Clabbers, Max T. B., et al.
(författare)
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Macromolecular crystallography using microcrystal electron diffraction
- 2021
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Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 77, s. 313-324
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Tidskriftsartikel (refereegranskat)abstract
- Microcrystal electron diffraction (MicroED) has recently emerged as a promising method for macromolecular structure determination in structural biology. Since the first protein structure was determined in 2013, the method has been evolving rapidly. Several protein structures have been determined and various studies indicate that MicroED is capable of (i) revealing atomic structures with charges, (ii) solving new protein structures by molecular replacement, (iii) visualizing ligand-binding interactions and (iv) determining membrane-protein structures from microcrystals embedded in lipidic mesophases. However, further development and optimization is required to make MicroED experiments more accurate and more accessible to the structural biology community. Here, we provide an overview of the current status of the field, and highlight the ongoing development, to provide an indication of where the field may be going in the coming years. We anticipate that MicroED will become a robust method for macromolecular structure determination, complementing existing methods in structural biology.
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| 14. |
- Clabbers, M. T. B., et al.
(författare)
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Protein structure determination by electron diffraction using a single three-dimensional nanocrystal
- 2017
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Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 73, s. 738-748
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Tidskriftsartikel (refereegranskat)abstract
- Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 mu m(3), i.e. no more than 6 x 10(5) unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 x 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.
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| 15. |
- Cotrim, Camila A., et al.
(författare)
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A structural overview of the zinc transporters in the cation diffusion facilitator family
- 2019
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Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 75, s. 357-367
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Forskningsöversikt (refereegranskat)abstract
- The cation diffusion facilitators (CDFs) are a family of membrane-bound proteins that maintain cellular homeostasis of essential metal ions. In humans, the zinc-transporter CDF family members (ZnTs) play important roles in zinc homeostasis. They do this by facilitating zinc efflux from the cytoplasm to the extracellular space across the plasma membrane or into intracellular organelles. Several ZnTs have been implicated in human health owing to their association with type 2 diabetes and neurodegenerative diseases. Although the structure determination of CDF family members is not trivial, recent advances in membrane-protein structural biology have resulted in two structures of bacterial YiiPs and several structures of their soluble C-terminal domains. These data reveal new insights into the molecular mechanism of ZnT proteins, suggesting a unique rocking-bundle mechanism that provides alternating access to the metal-binding site.
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| 16. |
- Daniel, Ed, et al.
(författare)
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IceBear : an intuitive and versatile web application for research-data tracking from crystallization experiment to PDB deposition
- 2021
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Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography (IUCr). - 2059-7983. ; 77:2, s. 151-163
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Tidskriftsartikel (refereegranskat)abstract
- The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank.
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| 17. |
- de la Rosa-Trevín, José Miguel, et al.
(författare)
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Development of basic building blocks for cryo-EM : the emcore and emvis software libraries
- 2020
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Ingår i: Acta Crystallographica Section D. - : INT UNION CRYSTALLOGRAPHY. - 2059-7983. ; 76, s. 350-356
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Tidskriftsartikel (refereegranskat)abstract
- Image-processing software has always been an integral part of structure determination by cryogenic electron microscopy (cryo-EM). Recent advances in hardware and software are recognized as one of the key factors in the so-called cryo-EM resolution revolution. Increasing computational power has opened many possibilities to consider more demanding algorithms, which in turn allow more complex biological problems to be tackled. Moreover, data processing has become more accessible to many experimental groups, with computations that used to last for many days at supercomputing facilities now being performed in hours on personal workstations. All of these advances, together with the rapid expansion of the community, continue to pose challenges and new demands on the software-development side. In this article, the development of emcore and emvis, two basic software libraries for image manipulation and data visualization in cryo-EM, is presented. The main goal is to provide basic functionality organized in modular components that other developers can reuse to implement new algorithms or build graphical applications. An additional aim is to showcase the importance of following established practices in software engineering, with the hope that this could be a first step towards a more standardized way of developing and distributing software in the field.
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| 18. |
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| 19. |
- Freitag-Pohl, Stefanie, et al.
(författare)
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Crystal structures of the Bacillus subtilis prophage lytic cassette proteins XepA and YomS.
- 2019
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 75, s. 1028-1039
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Tidskriftsartikel (refereegranskat)abstract
- As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPβ were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel β-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each β-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.
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| 20. |
- Griese, Julia J., et al.
(författare)
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Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion : Q-XAD
- 2019
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Ingår i: Acta Crystallographica Section D. - 2059-7983. ; D75, s. 764-771
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Tidskriftsartikel (refereegranskat)abstract
- Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.
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| 21. |
- Gudmundsson, Mikael, et al.
(författare)
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Structural and functional studies of the glycoside hydrolase family 3 beta-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii
- 2016
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Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 72:7, s. 860-870
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Tidskriftsartikel (refereegranskat)abstract
- The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. beta-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the beta-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the beta-glucosidase from H. jecorina (HjCel3A) with the beta-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 beta-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.
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| 22. |
- Hasse, Dirk, et al.
(författare)
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Structure and mechanism of piperideine-6-carboxylate dehydrogenase from Streptomyces clavuligerus
- 2019
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Ingår i: Acta Crystallographica Section D. - : INT UNION CRYSTALLOGRAPHY. - 2059-7983. ; 75, s. 1107-1118
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Tidskriftsartikel (refereegranskat)abstract
- The core of beta-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. beta-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid alpha-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form alpha-aminoadipate semialdehyde) to alpha-aminoadipic acid, is catalysed by the NAD(+)-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH from S. clavuligerus in its apo form and in complexes with the cofactor NAD(+), the product alpha-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD(+) binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.
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| 23. |
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| 24. |
- Jiang, Wangshu, et al.
(författare)
-
Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate
- 2019
-
Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography. - 2059-7983. ; 75, s. 618-627
-
Tidskriftsartikel (refereegranskat)abstract
- Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH 'locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 angstrom resolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised 'lock-and-trigger' mechanism for spider silk formation is presented.
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| 25. |
- Jiang, Wangshu, et al.
(författare)
-
Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis
- 2018
-
Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 74:Pt 11, s. 1053-1062
-
Tidskriftsartikel (refereegranskat)abstract
- The important uropathogen Proteus mirabilis encodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.5 Å resolution) and AtfE (1.58 Å resolution) from two P. mirabilis fimbriae (UCA/NAF and ATF) are presented. The structures of UcaD and AtfE are both similar to the F17G type of tip-located fimbrial receptor-binding domains, and the structures are very similar despite having only limited sequence similarity. These structures represent an important step towards a molecular-level understanding of P. mirabilis fimbrial adhesins and their roles in the complex pathogenesis of urinary-tract infections.
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| 26. |
- Kannan, K. K., et al.
(författare)
-
Bror Erik Strandberg (1930-2018)
- 2018
-
Ingår i: Acta Crystallographica Section D. - : International Union Of Crystallography. - 2059-7983. ; 74:7, s. 711-712
-
Tidskriftsartikel (populärvet., debatt m.m.)
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| 27. |
- Koruza, K, et al.
(författare)
-
Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX
- 2019
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 75:Pt 10, s. 895-903
-
Tidskriftsartikel (refereegranskat)abstract
- Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX-inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.
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| 28. |
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| 29. |
- Lima, Gustavo M A, et al.
(författare)
-
FragMAX : the fragment-screening platform at the MAX IV Laboratory
- 2020
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 76:Pt 8, s. 771-777
-
Tidskriftsartikel (refereegranskat)abstract
- Advances in synchrotron storage rings and beamline automation have pushed data-collection rates to thousands of data sets per week. With this increase in throughput, massive projects such as in-crystal fragment screening have become accessible to a larger number of research groups. The quality of support offered at large-scale facilities allows medicinal chemistry-focused or biochemistry-focused groups to supplement their research with structural biology. Preparing the experiment, analysing multiple data sets and prospecting for interesting complexes of protein and fragments require, for both newcomers and experienced users, efficient management of the project and extensive computational power for data processing and structure refinement. Here, FragMAX, a new complete platform for fragment screening at the BioMAX beamline of the MAX IV Laboratory, is described. The ways in which users are assisted in X-ray-based fragment screenings and in which the fourth-generation storage ring available at the facility is best exploited are also described.
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| 30. |
- Lima, Gustavo M.A., et al.
(författare)
-
FragMAXapp : Crystallographic fragment-screening data-analysis and project-management system
- 2021
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 799-808
-
Tidskriftsartikel (refereegranskat)abstract
- Crystallographic fragment screening (CFS) has become one of the major techniques for screening compounds in the early stages of drug-discovery projects. Following the advances in automation and throughput at modern macromolecular crystallography beamlines, the bottleneck for CFS has shifted from collecting data to organizing and handling the analysis of such projects. The complexity that emerges from the use of multiple methods for processing and refinement and to search for ligands requires an equally sophisticated solution to summarize the output, allowing researchers to focus on the scientific questions instead of on software technicalities. FragMAXapp is the fragment-screening project-management tool designed to handle CFS projects at MAX IV Laboratory. It benefits from the powerful computing infrastructure of large-scale facilities and, as a web application, it is accessible from everywhere.
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| 31. |
- Manne, Kartik, et al.
(författare)
-
Novel structure of the N-terminal helical domain of BibA, a group B streptococcus immunogenic bacterial adhesin
- 2020
-
Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 76, s. 759-770
-
Tidskriftsartikel (refereegranskat)abstract
- BibA, a group B streptococcus (GBS) surface protein, has been shown to protect the pathogen from phagocytic killing by sequestering a complement inhibitor: C4b-binding protein (C4BP). Here, the X-ray crystallographic structure of a GBS BibA fragment (BibA126-398) and a low-resolution small-angle X-ray scattering (SAXS) structure of the full-length N-terminal domain (BibA34-400) are described. The BibA126-398 fragment crystal structure displayed a novel and predominantly helical structure. The tertiary arrangement of helices forms four antiparallel three-helix-bundle-motif repeats, with one long helix from a bundle extending into the next. Multiple mutations on recombinant BibA34-400 delayed the degradation of the protein, and circular dichroism spectroscopy of BibA34-400 suggested a similar secondary-structure composition to that observed in the crystallized BibA126-398 fragment. A model was generated for the 92 N-terminal residues (BibA34-125) using structural similarity prediction programs, and a BibA34-400 model was generated by combining the coordinates of BibA34-126 and BibA126-398. The X-ray structure of BibA126-398 and the model of BibA34-400 fitted well into the calculated SAXS envelope. One possible binding site for the BibA N-terminal domain was localized to the N-terminal CCP (complement-control protein) domains of the C4BP α-chain, as indicated by the decreased binding of BibA to a ΔCCP1 C4BP α-chain mutant. In summary, it is suggested that the GBS surface protein BibA, which consists of three antiparallel α-helical-bundle motifs, is unique and belongs to a new class of Gram-positive surface adhesins.
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| 32. |
- Manzoni, Francesco, et al.
(författare)
-
Perdeuteration, crystallization, data collection and comparison of five neutron diffraction data sets of complexes of human galectin-3C
- 2016
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 72:11, s. 1194-1202
-
Tidskriftsartikel (refereegranskat)abstract
- Galectin-3 is an important protein in molecular signalling events involving carbohydrate recognition, and an understanding of the hydrogen-bonding patterns in the carbohydrate-binding site of its C-terminal domain (galectin-3C) is important for the development of new potent inhibitors. The authors are studying these patterns using neutron crystallography. Here, the production of perdeuterated human galectin-3C and successive improvement in crystal size by the development of a crystal-growth protocol involving feeding of the crystallization drops are described. The larger crystals resulted in improved data quality and reduced data-collection times. Furthermore, protocols for complete removal of the lactose that is necessary for the production of large crystals of apo galectin-3C suitable for neutron diffraction are described. Five data sets have been collected at three different neutron sources from galectin-3C crystals of various volumes. It was possible to merge two of these to generate an almost complete neutron data set for the galectin-3C-lactose complex. These data sets provide insights into the crystal volumes and data-collection times necessary for the same system at sources with different technologies and data-collection strategies, and these insights are applicable to other systems.Perdeuteration, purification and the growth of large crystals of the carbohydrate-recognition domain of galectin-3C are described. Five neutron diffraction data sets have been collected at four neutron sources; these are compared and two are merged.
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| 33. |
- Martiel, Isabelle, et al.
(författare)
-
Versatile microporous polymer-based supports for serial macromolecular crystallography
- 2021
-
Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 77, s. 1153-1167
-
Tidskriftsartikel (refereegranskat)abstract
- Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.
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| 34. |
- Mazurkewich, Scott, 1982, et al.
(författare)
-
Structural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylans
- 2023
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 79, s. 545-555
-
Tidskriftsartikel (refereegranskat)abstract
- In plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, Lf CE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of Lf CE15C and other CE15 family members with similar sequence characteristics.
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| 35. |
- Mazurkewich, Scott, 1982, et al.
(författare)
-
Structure of a C1/C4-oxidizing AA9 lytic polysaccharide monooxygenase from the thermophilic fungus Malbranchea cinnamomea
- 2021
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 1019-1026
-
Tidskriftsartikel (refereegranskat)abstract
- The thermophilic fungus Malbranchea cinnamomea contains a host of enzymes that enable its ability as an efficient degrader of plant biomass and that could be mined for industrial applications. This thermophilic fungus has been studied and found to encode eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), which collectively possess different substrate specificities for a range of plant cell-wall-related polysaccharides and oligosaccharides. To gain greater insight into the molecular determinants defining the different specificities, structural studies were pursued and the structure of McAA9F was determined. The enzyme contains the immunoglobulin-like fold typical of previously solved AA9 LPMO structures, but contains prominent differences in the loop regions found on the surface of the substrate-binding site. Most significantly, McAA9F has a broad substrate specificity, with activity on both crystalline and soluble polysaccharides. Moreover, it contains a small loop in a region where a large loop has been proposed to govern specificity towards oligosaccharides. The presence of the small loop leads to a considerably flatter and more open surface that is likely to enable the broad specificity of the enzyme. The enzyme contains a succinimide residue substitution, arising from intramolecular cyclization of Asp10, at a position where several homologous members contain an equivalent residue but cyclization has not previously been observed. This first structure of an AA9 LPMO from M. cinnamomea aids both the understanding of this family of enzymes and the exploration of the repertoire of industrially relevant lignocellulolytic enzymes from this fungus.
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| 36. |
- Mazurkewich, S., et al.
(författare)
-
Structure of a C1/C4-oxidizing AA9 lytic polysaccharide monooxygenase from the thermophilic fungus Malbranchea cinnamomea
- 2021
-
Ingår i: Acta Crystallographica Section D-Structural Biology. - : International Union of Crystallography (IUCr). - 2059-7983. ; 77, s. 1019-1026
-
Tidskriftsartikel (refereegranskat)abstract
- The thermophilic fungus Malbranchea cinnamomea contains a host of enzymes that enable its ability as an efficient degrader of plant biomass and that could be mined for industrial applications. This thermophilic fungus has been studied and found to encode eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), which collectively possess different substrate specificities for a range of plant cell-wall-related polysaccharides and oligosaccharides. To gain greater insight into the molecular determinants defining the different specificities, structural studies were pursued and the structure of McAA9F was determined. The enzyme contains the immunoglobulin-like fold typical of previously solved AA9 LPMO structures, but contains prominent differences in the loop regions found on the surface of the substrate-binding site. Most significantly, McAA9F has a broad substrate specificity, with activity on both crystalline and soluble polysaccharides. Moreover, it contains a small loop in a region where a large loop has been proposed to govern specificity towards oligosaccharides. The presence of the small loop leads to a considerably flatter and more open surface that is likely to enable the broad specificity of the enzyme. The enzyme contains a succinimide residue substitution, arising from intramolecular cyclization of Asp10, at a position where several homologous members contain an equivalent residue but cyclization has not previously been observed. This first structure of an AA9 LPMO from M. cinnamomea aids both the understanding of this family of enzymes and the exploration of the repertoire of industrially relevant lignocellulolytic enzymes from this fungus.
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| 37. |
- Polyakov, K. M., et al.
(författare)
-
Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water by Steccherinum murashkinskyi laccase : insights into the reaction mechanism
- 2017
-
Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography. - 2059-7983. ; 73:Pt 5, s. 388-401
-
Tidskriftsartikel (refereegranskat)abstract
- The laccase from Steccherinum murashkinskyi is a member of the large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates, accompanied by the reduction of dioxygen to water. The reducing properties of X-ray radiation and the high quality of the laccase crystals allow the study of the catalytic reduction of dioxygen to water directly in a crystal. A series of diffraction data sets with increasing absorbed radiation dose were collected from a single crystal of Steccherinum murashkinskyi laccase at 1.35 angstrom resolution. Changes in the active-site structure associated with the reduction of molecular oxygen to water on increasing the absorbed dose of ionizing radiation were detected. The structures in the series are mixtures of different states of the enzyme-substrate complex. Nevertheless, it was possible to interpret these structures as complexes of various oxygen ligands with copper ions in different oxidation states. The results allowed the mechanism of oxygen reduction catalyzed by laccases to be refined.
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| 38. |
- Ramos, Joao, et al.
(författare)
-
The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme
- 2021
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 1579-1590
-
Tidskriftsartikel (refereegranskat)abstract
- The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97-Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.
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| 39. |
- Rämisch, Sebastian, et al.
(författare)
-
Crystal structure of human chondroadherin : Solving a difficult molecular-replacement problem using de novo models
- 2017
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 73:1, s. 53-63
-
Tidskriftsartikel (refereegranskat)abstract
- Chondroadherin (CHAD) is a cartilage matrix protein that mediates the adhesion of isolated chondrocytes. Its protein core is composed of 11 leucine-rich repeats (LRR) flanked by cysteine-rich domains. CHAD makes important interactions with collagen as well as with cell-surface heparin sulfate proteoglycans and α2β1 integrins. The integrin-binding site is located in a region of hitherto unknown structure at the C-terminal end of CHAD. Peptides based on the C-terminal human CHAD (hCHAD) sequence have shown therapeutic potential for treating osteoporosis. This article describes a still-unconventional structure solution by phasing with de novo models, the first of a β-rich protein. Structure determination of hCHAD using traditional, though nonsystematic, molecular replacement was unsuccessful in the hands of the authors, possibly owing to a combination of low sequence identity to other LRR proteins, four copies in the asymmetric unit and weak translational pseudosymmetry. However, it was possible to solve the structure by generating a large number of de novo models for the central LRR domain using Rosetta and multiple parallel molecular-replacement attempts using AMPLE. The hCHAD structure reveals an ordered C-terminal domain belonging to the LRRCT fold, with the integrin-binding motif (WLEAK) being part of a regular α-helix, and suggests ways in which experimental therapeutic peptides can be improved. The crystal structure itself and docking simulations further support that hCHAD dimers form in a similar manner to other matrix LRR proteins.The structure of human chondroadherin (hCHAD), a 359-amino-acid protein with 11 leucine-rich repeats, has been solved by molecular replacement using de novo models of a 195-residue segment and the AMPLE pipeline. This demonstrates that even a fairly large β-rich protein is tractable to solution using de novo modelling. The structure of the C-terminal domain of hCHAD reveals the conformation of a medically relevant peptide.
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| 40. |
- Sharov, Grigory, et al.
(författare)
-
Using RELION software within the Scipion framework
- 2021
-
Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 77, s. 403-410
-
Tidskriftsartikel (refereegranskat)abstract
- Scipion is a modular image-processing framework that integrates several software packages under a unified interface while taking care of file formats and conversions. Here, new developments and capabilities of the Scipion plugin for the widely used RELION software package are presented and illustrated with an image-processing pipeline for published data. The user interfaces of Scipion and RELION are compared and the key differences are highlighted, allowing this manuscript to be used as a guide for both new and experienced users of this software. Different on-the-fly image-processing options are also discussed, demonstrating the flexibility of the Scipion framework.
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| 41. |
- Sprenger, Janina, et al.
(författare)
-
Guest-protein incorporation into solvent channels of a protein host crystal (hostal)
- 2021
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 471-485
-
Tidskriftsartikel (refereegranskat)abstract
- Soaking small molecules into the solvent channels of protein crystals is the most common method of obtaining crystalline complexes with ligands such as substrates or inhibitors. The solvent channels of some protein crystals are large enough to allow the incorporation of macromolecules, but soaking of protein guests into protein crystals has not been reported. Such protein host crystals (here given the name hostals) incorporating guest proteins may be useful for a wide range of applications in biotechnology, for example as cargo systems or for diffraction studies analogous to the crystal sponge method. The present study takes advantage of crystals of the Escherichia coli tryptophan repressor protein (ds-TrpR) that are extensively domain-swapped and suitable for incorporating guest proteins by diffusion, as they are robust and have large solvent channels. Confocal fluorescence microscopy is used to follow the migration of cytochrome c and fluorophore-labeled calmodulin into the solvent channels of ds-TrpR crystals. The guest proteins become uniformly distributed in the crystal within weeks and enriched within the solvent channels. X-ray diffraction studies on host crystals with high concentrations of incorporated guests demonstrate that diffraction limits of ∼2.5 Å can still be achieved. Weak electron density is observed in the solvent channels, but the guest-protein structures could not be determined by conventional crystallographic methods. Additional approaches that increase the ordering of guests in the host crystal are discussed that may support protein structure determination using the hostal system in the future. This host system may also be useful for biotechnological applications where crystallographic order of the guest is not required.
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| 42. |
- Sridhar, Shruthi, et al.
(författare)
-
Crystallographic binding studies of rat peroxisomal multifunctional enzyme type 1 with 3-ketodecanoyl-CoA : capturing active and inactive states of its hydratase and dehydrogenase catalytic sites
- 2020
-
Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography (IUCr). - 2059-7983. ; 76:12, s. 1256-1269
-
Tidskriftsartikel (refereegranskat)abstract
- The peroxisomal multifunctional enzyme type 1 (MFE1) catalyzes two successive reactions in the beta-oxidation cycle: the 2E-enoyl-CoA hydratase (ECH) and NAD(+)-dependent 3S-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 angstrom resolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD(+) bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part.
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| 43. |
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| 44. |
- Sørensen, Thomas Lykke Møller, et al.
(författare)
-
Membrane-protein crystals for neutron diffraction
- 2018
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 74:12, s. 1208-1218
-
Forskningsöversikt (refereegranskat)abstract
- Neutron macromolecular crystallography (NMX) has the potential to provide the experimental input to address unresolved aspects of transport mechanisms and protonation in membrane proteins. However, despite this clear scientific motivation, the practical challenges of obtaining crystals that are large enough to make NMX feasible have so far been prohibitive. Here, the potential impact on feasibility of a more powerful neutron source is reviewed and a strategy for obtaining larger crystals is formulated, exemplified by the calcium-transporting ATPase SERCA1. The challenges encountered at the various steps in the process from crystal nucleation and growth to crystal mounting are explored, and it is demonstrated that NMX-compatible membrane-protein crystals can indeed be obtained.
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| 45. |
- Valegård, Karin, et al.
(författare)
-
Structure of Rubisco from Arabidopsis thaliana in complex with 2-carboxyarabinitol-1,5-bisphosphate
- 2018
-
Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 74:1, s. 1-9
-
Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Arabidopsis thaliana is reported at 1.5 Å resolution. In light of the importance of A. thaliana as a model organism for understanding higher plant biology, and the pivotal role of Rubisco in photosynthetic carbon assimilation, there has been a notable absence of an A. thaliana Rubisco crystal structure. A. thaliana Rubisco is an L8S8 hexadecamer comprising eight plastome-encoded catalytic large (L) subunits and eight nuclear-encoded small (S) subunits. A. thaliana produces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B), and this crystal structure provides a snapshot of A. thaliana Rubisco containing the low-abundance RbcS3B small-subunit isoform. Crystals were obtained in the presence of the transition-state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate. A. thaliana Rubisco shares the overall fold characteristic of higher plant Rubiscos, but exhibits an interesting disparity between sequence and structural relatedness to other Rubisco isoforms. These results provide the structural framework to understand A. thaliana Rubisco and the potential catalytic differences that could be conferred by alternative A. thaliana Rubisco small-subunit isoforms.
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| 46. |
- Wallner, Björn
(författare)
-
Estimating local protein model quality: prospects for molecular replacement
- 2020
-
Ingår i: Acta Crystallographica Section D. - : INT UNION CRYSTALLOGRAPHY. - 2059-7983. ; 76, s. 285-290
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Tidskriftsartikel (refereegranskat)abstract
- Model quality assessment programs estimate the quality of protein models and can be used to estimate local error in protein models. ProQ3D is the most recent and most accurate version of our software. Here, it is demonstrated that it is possible to use local error estimates to substantially increase the quality of the models for molecular replacement (MR). Adjusting the B factors using ProQ3D improved the log-likelihood gain (LLG) score by over 50% on average, resulting in significantly more successful models in MR compared with not using error estimates. On a data set of 431 homology models to address difficult MR targets, models with error estimates from ProQ3D received an LLG of amp;gt;50 for almost half of the models 209/431 (48.5%), compared with 175/431 (40.6%) for the previous version, ProQ2, and only 74/431 (17.2%) for models with no error estimates, clearly demonstrating the added value of using error estimates to enable MR for more targets. ProQ3D is available from http://proq3.bioinfo.se/ both as a server and as a standalone download.
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