Murray M.S.,Rigaku Americas Corporation |
Milliner D.S.,Mayo Clinic Hyperoxaluria Center
Biochimica et Biophysica Acta - Molecular Basis of Disease | Year: 2012
Mutations in the gene encoding for 4-hydroxy-2-oxoglutarate aldolase (HOGA) are associated with an excessive production of oxalate in Primary Hyperoxaluria type 3 (PH3). This enzyme is the final step of the hydroxyproline degradation pathway within the mitochondria and catalyzes the cleavage of 4-hydroxy-2-oxoglutarate (HOG) to pyruvate and glyoxylate. No analyses have been performed to assess the consequences of the mutations identified, particularly for those variants that produce either full-length or nearly full-length proteins. In this study, the expression, stability, and activity of nine PH3 human HOGA variants were examined. Using recombinant protein produced in Escherichia coli as well as transfected Chinese hamster ovary (CHO) cells, it was found that all nine PH3 variants are quite unstable, have a tendency to aggregate, and retain no measurable activity. A buildup of HOG was confirmed in the urine, sera and liver samples from PH3 patients. To determine how HOG is cleaved in the absence of HOGA activity, the ability of N-acetylneuraminate aldolase (NAL) to cleave HOG was evaluated. NAL showed minimal activity towards HOG. Whether the expected buildup of HOG in mitochondria could inhibit glyoxylate reductase (GR), the enzyme mutated in PH2, was also evaluated. GR was inhibited by HOG but not by 2-hydroxyglutarate or 2-oxoglutarate. Thus, one hypothetical component of the molecular basis for the excessive oxalate production in PH3 appears to be the inhibition of GR by HOG, resulting in a phenotype similar to PH2. © 2012 Elsevier B.V.
Cherry A.L.,Karolinska Institutet |
Finta C.,Karolinska Institutet |
Karlstrom M.,Karolinska Institutet |
Jin Q.,Karolinska Institutet |
And 9 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013
Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics. © 2013 International Union of Crystallography.
News Article | February 23, 2017
Rigaku Americas Corporation has published a new comprehensive catalog featuring its current portfolio of X-ray analytical instrumentation. Rigaku provides a complete line of analytical and industrial instrumentation covering the full spectrum of X-ray based test and measurement equipment. The new publication presents the company’s entire line of materials analysis systems for the North and South American markets. The catalog enumerates the company’s X-ray Diffraction (XRD), Wavelength and Energy Dispersive X-ray Fluorescence (XRF), Small Angle X-ray scattering (SAXS) and X-ray Computed Microtomography (μCT) systems. Both full-sized laboratory systems and portable/benchtop systems for use in the field are included. Analytical techniques enabled by Rigaku technology are suitable for performing rapid non-destructive elemental analysis in all types of samples across a range of industries. Overviews of the principles of the instruments’ respective technologies are presented along with descriptions of the unique capabilities of each system, indicating that Rigaku is able to offer an optimized solution for virtually any kind of X-ray analytical task. A catalog can be requested at info(at)rigaku(dot)com. About Rigaku Since its inception in Japan in 1951, Rigaku has been at the forefront of analytical and industrial instrumentation technology. Rigaku and its subsidiaries form a global group focused on general-purpose analytical instrumentation and the life sciences. With hundreds of major innovations to their credit, Rigaku companies are world leaders in X-ray spectrometry, diffraction, and optics, as well as small molecule and protein crystallography and semiconductor metrology. Today, Rigaku employs over 1,400 people in the manufacturing and support of its analytical equipment, which is used in more than 90 countries around the world supporting research, development, and quality assurance activities. Throughout the world, Rigaku continuously promotes partnerships, dialog, and innovation within the global scientific and industrial communities.
Polizzi S.J.,University of Georgia |
Walsh Jr. R.M.,University of Georgia |
Le Magueres P.,Rigaku Americas Corporation |
Criswell A.R.,Rigaku Americas Corporation |
Wood Z.A.,University of Georgia
Biochemistry | Year: 2013
Human UDP-α-d-xylose synthase (hUXS) is a member of the extended short chain dehydrogenase/reductase (SDR) family of enzymes. Previous crystallographic studies have shown that hUXS conserves the same dimeric quaternary structure observed in other SDR enzymes. Here, we present evidence that hUXS also forms a tetramer in solution that is important for activity. Sedimentation velocity studies show that two hUXS dimers undergo a concentration-dependent association to form a tetramer with a Kd of 2.9 μM. The tetrameric complex is also observed in small-angle X-ray scattering (SAXS). The specific activity for the production of the reaction intermediate UDP-α-d-4-keto-xylose displays a hyperbolic dependence on protein concentration that is well modeled by an isotherm using the 2.9 μM Kd of the tetramer. Likewise, the rate of UDP-α-d-xylose production in the presence of increasing concentrations of the small molecule crowder trimethylamine N-oxide is consistent with the formation of a higher activity tetramer. We present several possible structural models of the hUXS tetramer based on (i) hUXS crystal packing, (ii) homology modeling, or (iii) ab initio simulated annealing of dimers. We analyze the models in terms of packing quality and agreement with SAXS data. The higher activity of the tetramer coupled with the relative instability of the complex suggests that an association-dissociation mechanism may regulate hUXS activity. © 2013 American Chemical Society.
Jaeger A.M.,Duke University |
Pemble C.W.,Duke University |
Pemble C.W.,Rigaku Americas Corporation |
Sistonen L.,Åbo Akademi University |
Thiele D.J.,Duke University
Nature Structural and Molecular Biology | Year: 2016
Heat-shock transcription factor (HSF) family members function in stress protection and in human diseases including proteopathies, neurodegeneration and cancer. The mechanisms that drive distinct post-translational modifications, cofactor recruitment and target-gene activation for specific HSF paralogs are unknown. We present crystal structures of the human HSF2 DNA-binding domain (DBD) bound to DNA, revealing an unprecedented view of HSFs that provides insights into their unique biology. The HSF2 DBD structures resolve a new C-terminal helix that directs wrapping of the coiled-coil domain around DNA, thereby exposing paralog-specific sequences of the DBD surface for differential post-translational modifications and cofactor interactions. We further demonstrate a direct interaction between HSF1 and HSF2 through their coiled-coil domains. Together, these features provide a new model for HSF structure as the basis for differential and combinatorial regulation, which influences the transcriptional response to cellular stress. © 2016 Nature America, Inc.
Rajasekaran D.,Yale University |
Fan C.,Yale University |
Meng W.,Yale University |
Pflugrath J.W.,Rigaku Americas Corporation |
Lolis E.J.,Yale University
Proteins: Structure, Function and Bioinformatics | Year: 2014
The mammalian chemokine family is segregated into four families - CC, CXC, CX3C, and XC-based on the arrangement of cysteines and the corresponding disulfides. Sequencing of the Danio rerio (zebrafish) genome has identified more than double the amount of human chemokines with the absence of the CX3C family and the presence of a new family, CX. The only other family with a single cysteine in the N-terminal region is the XC family. Human lymphotactin (XCL1) has two interconverting structures due to dynamic changes that occur in the protein. Similar to an experiment with XCL1 that identified the two structural forms, we probed for multiple forms of zCXL1 using heparin affinity. The results suggest only a single form of CXL1 is present. We used sulfur-SAD phasing to determine the three-dimensional structure CXL1. Zebrafish CXL1 (zCXL1) has three disulfides that appear to be important for a stable structure. One disulfide is common to all chemokines except those that belong to the XC family, another is similar to a subset of CC chemokines containing three disulfides, but the third disulfide is unique to the CX family. We analyzed the electrostatic potential of the zCXL1 structure and identified the likely heparin-binding site for glycosaminoglycans (GAGs). zCXL1 has a similar sequence identity with human CCL5 and CXCL12, but the structure is more related to CCL5. Our structural analysis supports the phylogenetic and genomic studies on the evolution of the CXL family. © 2013 Wiley Periodicals, Inc.
Criswell A.,Rigaku Americas Inc |
Mooers B.H.M.,The University of Oklahoma Health Sciences Center
Methods in Molecular Biology | Year: 2015
We used small-angle X-ray scattering (SAXS) to evaluate the solution structure of a double-stranded RNA with 32 base pairs. We wanted to compare the solution structure to the crystal structure to assess the impact of the crystal lattice on the overall conformation of the RNA. The RNA was designed to self-anneal and form a head-to-head fusion of two identical mRNA/oligo(U) tail domains (the U-helix) from a trypanosome RNA editing substrate formed by the annealing of a guide RNA to a pre-edited mRNA. This substrate is from the U insertion/deletion RNA editing system of trypanosomes. Each strand in the fusion RNA had 16 purines from the pre-mRNA followed by 16 uracils (Us) from the U-tail at the 3′ end of the guide RNA. The strands were designed to form a double helix with blunt ends, but each strand had the potential to form hairpins and single-stranded RNA helices. Hairpins could form by the 3′ oligouridylate tract folding back to hybridize with the 5′ oligopurine tract and forming an intervening loop. Single-stranded helices could form by the stacking of bases in the polypurine tract. Some of the 16 Us 3′ to the polypurine tract may have been unstacked and in random coils. Our SAXS studies showed that the RNA formed a mix of single-stranded structures in the absence of MgCl2. In the presence of MgCl2 at concentrations similar to those in the crystal, the solution structure was consistent with the double- stranded, blunt-ended structure, in agreement with the crystal structure. Here we describe the preparation of RNA samples, data collection with an in-house SAXS instrument designed for biological samples, and the processing and modeling of the scattering data. © Springer Science+Business Media New York 2015.
Pflugrath J.W.,Rigaku Americas Corporation
Acta Crystallographica Section F:Structural Biology Communications | Year: 2015
Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented. © 2015 International Union of Crystallography.
Rigaku Americas Corporation | Date: 2010-12-17
Imaging apparatus and instruments for use in the study of proteins and peptides in drug development.
Rigaku Americas Corporation | Date: 2014-11-26
Scientific instruments in the nature of x-ray diffraction (XRD) analysis instruments with advanced software control that provide materials analysis measurements of thin film materials in such fields as semiconductor manufacturing and soil analysis.