| 1. |
- Zhou, Yan-Feng, et al.
(författare)
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C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 383:1, s. 49-61
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Tidskriftsartikel (refereegranskat)abstract
- C(4)-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C(4)-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C(4)-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C(4) succinate, and a complex with C(3) malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C(4)-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- Brostromer, Erik, et al.
(författare)
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An automated image-collection system for crystallization experiments using SBS standard microplates
- 2007
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 0907-4449. ; 63:Pt 2, s. 25-119
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Tidskriftsartikel (refereegranskat)abstract
- As part of a structural genomics platform in a university laboratory, a low-cost in-house-developed automated imaging system for SBS microplate experiments has been designed and constructed. The imaging system can scan a microplate in 2-6 min for a 96-well plate depending on the plate layout and scanning options. A web-based crystallization database system has been developed, enabling users to follow their crystallization experiments from a web browser. As the system has been designed and built by students and crystallographers using commercially available parts, this report is aimed to serve as a do-it-yourself example for laboratory robotics.
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| 5. |
- Brostromer, Erik, et al.
(författare)
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Solid-liquid interface method (SLIM) : a new crystallization method for proteins
- 2009
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 386:4, s. 8-634
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Tidskriftsartikel (refereegranskat)abstract
- Despite impressive advances in theories, methods and technologies, crystallization still remains a serious bottleneck in structural determination of macromolecules. Here we present a novel solid-liquid interface method (SLIM) for protein crystallization, based on the pre-adding and drying of a crystallization reagent, and thereafter the dispensing of a protein solution to the dried media to initiate crystallization from the solid-liquid interface. Not only quick and easy to perform, the method also allows for a less concentrated protein solution for setting up crystallization trials.
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| 6. |
- Chen, Hanwei, et al.
(författare)
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Tumor Volumes Measured From Static and Dynamic F-18-fluoro-2-deoxy-D-glucose Positron Emission Tomography-Computed Tomography Scan : Comparison of Different Methods Using Magnetic Resonance Imaging as the Criterion Standard
- 2014
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Ingår i: Journal of computer assisted tomography. - 0363-8715 .- 1532-3145. ; 38:2, s. 209-215
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Tidskriftsartikel (refereegranskat)abstract
- Objective: The objective of this study was to compare the accuracy of calculating the primary tumor volumes using a gradient-based method and fixed threshold methods on the standardized uptake value (SUV) maps and the net influx of FDG (Ki) maps from positron emission tomography-computed tomography (PET-CT) images. Materials and Methods: Newly diagnosed patients with head and neck cancer were recruited, and dynamic PET-CT scan and T2-weighted magnetic resonance imaging were performed. The maps of Ki and SUV were calculated from PET-CT images. The tumor volumes were calculated using a gradient-based method and a fixed threshold method at 40% of maximal SUV or maximal Ki. Four kinds of volumes, VOLKi-Gra (from the Ki maps using the gradient-based method), VOLKi-40% (from the Ki maps using the threshold of 40% maximal Ki), VOLSUV-Gra (from the SUV maps using the gradient-based method), and VOLSUV-40% (from the SUV maps using the threshold of 40% maximal SUV), were acquired and compared with VOLMRI (the volumes acquired on T2-weighted images) using the Pearson correlation, paired t test, and similarity analysis. Results: Eighteen patients were studied, of which 4 had poorly defined tumors (PDT). The positron emission tomography-derived volumes were as follows: VOLSUV-40%, 2.1 to 41.2 cm(3) (mean [SD], 12.3 [10.6]); VOLSUV-Gra, 2.2 to 28.1 cm(3) (mean [SD], 13.2 [8.4]); VOLKi-Gra, 2.4 to 17.0 cm(3) (mean [SD], 9.5 [4.6]); and VOLKi-40%, 2.7 to 20.3 cm(3) (mean [SD], 12.0 [6.0]). The VOLMRI ranged from 2.9 to 18.1 cm(3) (mean [SD], 9.1 [3.9]). The VOLKi-Gra significantly correlated with VOLMRI with the highest correlation coefficient (PDT included, R = 0.673, P = 0.002; PDT excluded, R = 0.841, P < 0.001) and presented no difference from VOLMRI (P = 0.672 or 0.561, respectively, PDT included and excluded). The difference between VOLKi-Gra and VOLMRI was also the smallest. Conclusions: The tumor volumes delineated on the Ki maps using the gradient-based method are more accurate than those on the SUV maps and using the fixed threshold methods.
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| 7. |
- Chen, Mei-Qin, et al.
(författare)
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Arabidopsis NMD3 is required for nuclear export of 60S ribosomal subunits and affects secondary cell wall thickening
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:4, s. 35904-35904
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Tidskriftsartikel (refereegranskat)abstract
- NMD3 is required for nuclear export of the 60S ribosomal subunit in yeast and vertebrate cells, but no corresponding function of NMD3 has been reported in plants. Here we report that Arabidopsis thaliana NMD3 (AtNMD3) showed a similar function in the nuclear export of the 60S ribosomal subunit. Interference with AtNMD3 function by overexpressing a truncated dominant negative form of the protein lacking the nuclear export signal sequence caused retainment of the 60S ribosomal subunits in the nuclei. More interestingly, the transgenic Arabidopsis with dominant negative interference of AtNMD3 function showed a striking failure of secondary cell wall thickening, consistent with the altered expression of related genes and composition of cell wall components. Observation of a significant decrease of rough endoplasmic reticulum (RER) in the differentiating interfascicular fiber cells of the transgenic plant stems suggested a link between the defective nuclear export of 60S ribosomal subunits and the abnormal formation of the secondary cell wall. These findings not only clarified the evolutionary conservation of NMD3 functions in the nuclear export of 60S ribosomal subunits in yeast, animals and plants, but also revealed a new facet of the regulatory mechanism underlying secondary cell wall thickening in Arabidopsis. This new facet is that the nuclear export of 60S ribosomal subunits and the formation of RER may play regulatory roles in coordinating protein synthesis in cytoplasm and transcription in nuclei.
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| 8. |
- Digre, Andreas, et al.
(författare)
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Heparin interactions with apoA1 and SAA in inflammation-associated HDL
- 2016
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 474:2, s. 309-314
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein A1 (apoA1) is the main protein component responsible for transportation of cholesterol on high-density lipoprotein (HDL). Serum amyloid A (SAA) is an acute phase protein associated with HDL. Apart from their physiological functions, both apoA1 and SAA have been identified as 'amyloidogenic peptides'. We report herein that the polysaccharide heparin interacts with both apoA1 and SAA in HDL isolated from plasma of inflamed mice. The reaction is rapid, forming complex aggregates composed of heparin, apoA1 and SAA as revealed by gel electrophoresis. This interaction is dependent on the size and concentration of added heparin. Mass spectrometry analysis of peptides derived from chemically crosslinked HDL-SAA particles detected multiple crosslinks between apoA1 and SAA, indicating close proximity (within 25 angstrom) of these two proteins on the HDL surface, providing a molecular and structural mechanism for the simultaneous binding of heparin to apoA1 and SAA.
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| 9. |
- Dong, Jingran, et al.
(författare)
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Kinetics and mechanism of oxidation of the anti-tubercular prodrug isoniazid and its analog by iridium(IV) as models for biological redox systems
- 2017
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Ingår i: Dalton Transactions. - : Royal Society of Chemistry (RSC). - 1477-9234 .- 1477-9226. ; 46:26, s. 8377-8386
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Tidskriftsartikel (refereegranskat)abstract
- A complex reaction mechanism of oxidation of the anti-tubercular prodrug isoniazid (isonicotinic hydrazide, INH) by [IrCl6]2− as a model for redox processes of such drugs in biological systems has been studied in aqueous solution as a function of pH between 0 and 8.5. Similar experiments have been performed with its isomer nicotinic hydrazide (NH). All reactions are overall second-order, first-order in [IrCl6]2− and hydrazide, and the observed second-order rate constants k′ have been determined as a function of pH. Spectrophotometric titrations indicate a stoichiometry of [Ir(IV)]:[hydrazide] = 4:1. HPLC analysis shows that the oxidation product of INH is isonicotinic acid. The derived reaction mechanism, based on rate law, time-resolved spectra and stoichiometry, involves parallel attacks by [IrCl6]2− on all four protolytic species of INH and NH as rate-determining steps, depending on pH. These steps are proposed to generate two types of hydrazyl free radicals. These radicals react further in three rapid consecutive processes, leading to the final oxidation products. Rate constants for the rate-determining steps have been determined for all protolytic species I–IV of INH and NH. They are used to calculate reactivity–pH diagrams. These diagrams demonstrate that for both systems, species IV is ca. 105 times more reactive in the redox process than the predominant species III at the physiological pH of 7.4. Thus, species IV will be the main reactant, in spite of the fact that its concentration at this pH is extremely low, a fact that has not been considered in previous work. The results indicate that pH changes might be an important factor in the activation process of INH in biological systems also, and that in such systems this process most likely is more complicated than previously assumed
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| 10. |
- Duan, Ming-Rui, et al.
(författare)
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DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 35:4, s. 54-1145
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Tidskriftsartikel (refereegranskat)abstract
- WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 A resolution has revealed that this domain is composed of a globular structure with five beta strands, forming an antiparallel beta-sheet. A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at beta2 and beta3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.
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| 11. |
- Ebhardt, H Alexander, et al.
(författare)
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Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage
- 2014
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Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 1097-0231 .- 0951-4198. ; 28:24, s. 43-2735
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Tidskriftsartikel (refereegranskat)abstract
- RATIONALE: Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues.METHODS: Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNA(Arg) onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini.RESULTS: We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue.CONCLUSIONS: We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern.
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| 12. |
- Fu, Tian-Min, et al.
(författare)
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Protein preparation, crystallization and preliminary X-ray analysis of the C-terminal domain of human RSK1 serine/threonine protein kinase
- 2007
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Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications. - 2053-230X. ; 63:Pt 12, s. 8-1026
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Tidskriftsartikel (refereegranskat)abstract
- As a substrate of extracellular signal-related kinase (ERK), the p90 ribosome S6 kinase 1 (RSK1) is at the terminus of the Ras/ERK pathway. Residues 411-735 of human RSK1, covering the C-terminal serine/threonine kinase catalytic domain and the functionally important tail, were cloned into an Escherichia coli expression vector. The protein was expressed, purified and crystallized. The crystals diffracted to 2.7 A and belonged to space group P2(1), with unit-cell parameters a = 39.8, b = 143.8, c = 59.9 A, beta = 95.7 degrees.
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| 13. |
- Gao, Yun, et al.
(författare)
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Region separation type bio-photoelectrode based all-solid-state self-powered aptasensor for ochratoxin A and aflatoxin B1 detection
- 2022
-
Ingår i: Sensors and actuators. B, Chemical. - : Elsevier. - 0925-4005 .- 1873-3077. ; 364
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Tidskriftsartikel (refereegranskat)abstract
- Ochratoxin A (OTA) and Aflatoxin B1 (AFB1) are two highly toxic and naturally coexistence mycotoxins, which have posed a serious threat to food safety. The coexistence of these two mycotoxins can produce significant synergistic effects, so it is necessary to establish an effective analytical method. In this work, a dual biophotoelectrode based all-solid-state multiplexed self-powered aptasensor was realized for high-throughput analysis of OTA and AFB1. There was a large Fermi level difference between photoanode and photocathode, which ensured the light assisted self-driving of the system. Due to the regional immobilization of aptamers, it could save the dosage of recognition elements, reduce the preparation cost and simplify the operation procedure. Meanwhile, construction strategy of spatial separation could effectively eliminate the overlapping signals and cross interference between targets, achieving the results more accurate and the calculation more convenient. The constructed aptasensor realized OTA and AFB1 detection in corn samples with practicality, good stability, antiinterference ability and repeatability. Therefore, this work not only achieved the high-throughput analysis of mycotoxins, but also provided a new perspective for the construction of all-solid-state multiplexed self-powered sensor.
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| 14. |
- Ghosh, Swagatha, 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. - 0021-8898. ; 56:Pt 2, s. 449-460
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Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography was initially developed for room-temperature 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 goniometer-compatible 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 Å resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.
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| 15. |
- Gorgisyan, Ishkhan, et al.
(författare)
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Fast, automated, continuous energy scans for experimental phasing at the BioMAX beamline
- 2023
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Ingår i: Journal of Synchrotron Radiation. - 0909-0495. ; 30, s. 885-894
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Tidskriftsartikel (refereegranskat)abstract
- In X-ray macromolecular crystallography (MX), single-wavelength anomalous dispersion (SAD) and multi-wavelength anomalous dispersion (MAD) techniques are commonly used for obtaining experimental phases. For an MX synchrotron beamline to support SAD and MAD techniques it is a prerequisite to have a reliable, fast and well automated energy scan routine. This work reports on a continuous energy scan procedure newly implemented at the BioMAX MX beamline at MAX IV Laboratory. The continuous energy scan is fully automated, capable of measuring accurate fluorescence counts over the absorption edge of interest while minimizing the sample exposure to X-rays, and is about a factor of five faster compared with a conventional step scan previously operational at BioMAX. The implementation of the continuous energy scan facilitates the prompt access to the anomalous scattering data, required for the SAD and MAD experiments.
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| 16. |
- Leonarski, Filip, et al.
(författare)
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Kilohertz serial crystallography with the JUNGFRAU detector at a fourth-generation synchrotron source
- 2023
-
Ingår i: IUCrJ. - 2052-2525. ; 10:Pt 6, s. 729-737
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Tidskriftsartikel (refereegranskat)abstract
- Serial and time-resolved macromolecular crystallography are on the rise. However, beam time at X-ray free-electron lasers is limited and most thirdgeneration synchrotron-based macromolecular crystallography beamlines do not offer the necessary infrastructure yet. Here, a new setup is demonstrated, based on the JUNGFRAU detector and Jungfraujoch data-acquisition system, that enables collection of kilohertz serial crystallography data at fourthgeneration synchrotrons. More importantly, it is shown that this setup is capable of collecting multiple-time-point time-resolved protein dynamics at kilohertz rates, allowing the probing of microsecond to second dynamics at synchrotrons in a fraction of the time needed previously. A high-quality complete X-ray dataset was obtained within 1 min from lysozyme microcrystals, and the dynamics of the light-driven sodium-pump membrane protein KR2 with a time resolution of 1 ms could be demonstrated. To make the setup more accessible for researchers, downstream data handling and analysis will be automated to allow on-the-fly spot finding and indexing, as well as data processing.
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| 17. |
- Li, Gui-Lan, et al.
(författare)
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Open-closed conformational change revealed by the crystal structures of 3-keto-L-gulonate 6-phosphate decarboxylase from Streptococcus mutans
- 2009
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 381:3, s. 33-429
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Tidskriftsartikel (refereegranskat)abstract
- The 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC) catalyses the decarboxylation of 3-keto-L-gulonate 6-phosphate to L-xylulose in the presence of magnesium ions. The enzyme is involved in L-ascorbate metabolism and plays an essential role in the pathway of glucuronate interconversion. Crystal structures of Streptococcus mutans KGPDC were determined in the absence and presence of the product analog D-ribulose 5-phosphate. We have observed an 8 A alphaB-helix movement and other structural rearrangements around the active site between the apo-structures and product analog bound structure. These drastic conformational changes upon ligand binding are the first observation of this kind for the KGPDC family. The flexibilities of both the alpha-helix lid and the side chains of Arg144 and Arg197 are associated with substrate binding and product releasing. The open-closed conformational changes of the active site, through the movements of the alpha-helix lid and the arginine residues are important for substrate binding and catalysis.
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| 18. |
- Li, Lanfen, et al.
(författare)
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Structural genomics studies of human caries pathogen Streptococcus mutans
- 2014
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Ingår i: Journal of Structural and Functional Genomics. - : Springer Science and Business Media LLC. - 1345-711X .- 1570-0267. ; 15:3, s. 9-91
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Tidskriftsartikel (refereegranskat)abstract
- Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.
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| 19. |
- Logan, Derek, et al.
(författare)
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Status of the crystallography beamlines at the MAX IV Laboratory
- 2015
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Ingår i: The European Physical Journal Plus. - : Springer Science and Business Media LLC. - 2190-5444. ; 130:3
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Forskningsöversikt (refereegranskat)abstract
- The MAX IV Laboratory in Lund is currently operating two storage rings, the 1.5 GeV MAX II and the 700MeV MAX III, as well as constructing the new facility MAX IV, which will house a 1.5 GeV and a 3 GeV ring. At the MAX II synchrotron there are three hard X-ray beamlines at which crystallography can be performed: I711, I811 and I911. Beamline I711 is mainly used for powder diffraction. I811 is an EXAFS station at which surface XRD can also be carried out. I911 is a beamline with five experimental stations on a single superconducting wiggler source, of which two are currently used for macromolecular crystallography, namely the monochromatic station I911-2 and the tuneable station I911-3, which is equipped with a state-of-the-art goniometer and robotic sample changer. We will give an overview of the capabilities of these beamlines, focusing particularly on the macromolecular crystallography beamline I911 and some recent scientific highlights produced there. We will also give a brief overview of new beamlines for crystallography that are under construction or planned for the MAX IV facility.
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| 20. |
- Lu, Lu, et al.
(författare)
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Crystal structure of tubulin folding cofactor A from Arabidopsis thaliana and its beta-tubulin binding characterization
- 2010
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 584:16, s. 9-3533
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Tidskriftsartikel (refereegranskat)abstract
- Microtubules are composed of polymerized alpha/beta-tubulin heterodimers. Biogenesis of assembly-competent tubulin dimers is a complex multistep process that requires sequential actions of distinct molecular chaperones and cofactors. Tubulin folding cofactor A (TFCA), which captures beta-tubulin during the folding pathway, has been identified in many organisms. Here, we report the crystal structure of Arabidopsis thaliana TFC A (KIESEL, KIS), which forms a monomeric three-helix bundle. The functional binding analysis demonstrated that KIS interacts with beta-tubulin in plant. Furthermore, mutagenesis studies indicated that the alpha-helical regions of KIS participate in beta-tubulin binding. Unlike the budding yeast TFC A, the two loop regions of KIS are not required for this interaction suggesting a distinct binding mechanism of TFC A to beta-tubulin in plants.
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| 21. |
- Lu, Lu, et al.
(författare)
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Crystallization and preliminary X-ray analysis of tubulin-folding cofactor A from Arabidopsis thaliana
- 2010
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Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications. - 2053-230X. ; 66:Pt 8, s. 6-954
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Tidskriftsartikel (refereegranskat)abstract
- Tubulin-folding cofactor A (TFC A) is a molecular post-chaperonin that is involved in the beta-tubulin-folding pathway. It has been identified in many organisms including yeasts, humans and plants. In this work, Arabidopsis thaliana TFC A was expressed in Escherichia coli and purified to homogeneity. After thrombin cleavage, a well diffracting crystal was obtained by the sitting-drop vapour-diffusion method at 289 K. The crystal diffracted to 1.6 A resolution using synchrotron radiation and belonged to space group I4(1), with unit-cell parameters a=55.0, b=55.0, c=67.4 A.
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| 22. |
- Müller, Uwe, et al.
(författare)
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MXCuBE3 : A New Era of MX-Beamline Control Begins
- 2017
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Ingår i: Synchrotron Radiation News. - : Informa UK Limited. - 0894-0886 .- 1931-7344. ; 30:1, s. 22-27
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Tidskriftsartikel (refereegranskat)abstract
- The outstanding success of structural biology within the last two decades is closely related to the development and evolution of macromolecular crystallography (MX) beamlines. Indeed, many of today's synchrotron-based MX experimental sessions aim for fast but rigorous evaluations and data collections from very large numbers of samples [1–7]. To facilitate this, sample changing on most MX beamlines is now carried out by robots and the centering of a crystal in the X-ray beam to micrometer precision is now automatically performed using either optical or diffraction-based techniques [8]. Once a crystal is centered, users have a wide array of options at their disposal to prepare any given experiment. This includes: X-ray fluorescence (XRF) [9] analysis to confirm the presence of anomalous scatterers in crystals; X-ray absorption near-edge scans (XANES) to determine the best X-ray wavelengths for MAD/SAD data collection [10]; and the probing of the diffraction properties of crystals to determine the best crystal, or area of a crystal [11], for data collection. All of these operations are now also automated, as is the collection of the final diffraction data set either from single or multiple crystals and the subsequent data analysis and reduction.
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| 23. |
- Nan, Jie, et al.
(författare)
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Bioinformatics and structural characterization of a hypothetical protein from Streptococcus mutans : implication of antibiotic resistance
- 2009
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:10, s. 7245-7245
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Tidskriftsartikel (refereegranskat)abstract
- As an oral bacterial pathogen, Streptococcus mutans has been known as the aetiologic agent of human dental caries. Among a total of 1960 identified proteins within the genome of this organism, there are about 500 without any known functions. One of these proteins, SMU.440, has very few homologs in the current protein databases and it does not fall into any protein functional families. Phylogenetic studies showed that SMU.440 is related to a particular ecological niche and conserved specifically in some oral pathogens, due to lateral gene transfer. The co-occurrence of a MarR protein within the same operon among these oral pathogens suggests that SMU.440 may be associated with antibiotic resistance. The structure determination of SMU.440 revealed that it shares the same fold and a similar pocket as polyketide cyclases, which indicated that it is very likely to bind some polyketide-like molecules. From the interlinking structural and bioinformatics studies, we have concluded that SMU.440 could be involved in polyketide-like antibiotic resistance, providing a better understanding of this hypothetical protein. Besides, the combination of multiple methods in this study can be used as a general approach for functional studies of a protein with unknown function.
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| 24. |
- Nan, Jie, et al.
(författare)
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Structure of a fatty-acid-binding protein from Bacillus subtilis determined by sulfur-SAD phasing using in-house chromium radiation
- 2009
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; 65:Pt 5, s. 8-440
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Tidskriftsartikel (refereegranskat)abstract
- Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for ab initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5 A resolution using Cr Kalpha radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family.
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| 25. |
- Oscarsson, Markus, et al.
(författare)
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MXCuBE2: the dawn of MXCuBE Collaboration
- 2019
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Ingår i: Journal of Synchrotron Radiation. - 1600-5775.
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Tidskriftsartikel (refereegranskat)abstract
- MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF – the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community. © Marcus Oscarsson et al. 2019
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