<i>BioXTAS RAW</i>: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis

Hopkins, Jesse B.; Gillilan, Richard E.; Skou, Søren

doi:10.1107/s1600576717011438

Cited by 546 publications

(495 citation statements)

References 66 publications

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“…All SAXS experiments were collected at the Cornell High-Energy Synchrotron Source (CHESS)’s G1 beamline using a dual Pilatus 100K-S SAXS/WAXS detector. 32,33 Capillary cells were robotically loaded with the 30 μL pre-centrifuged samples, maintained at 4°C, and thoroughly washed between samples. 34 For 0.005 mM, 0.01 mM and 0.03 mM concentrations of 11m , each sample condition and SAXS buffer was loaded and measured as three 30 μL replicates.…”

Section: Methodsmentioning

confidence: 99%

7-Phenoxy-Substituted 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides as Positive Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors with Nanomolar Potency

et al. 2017

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We report here the synthesis of 7-phenoxy-substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides and their evaluation as AMPA receptor positive allosteric modulators (AMPApams). The impact of substitution on the phenoxy ring and on the nitrogen atom at the 4-position was examined. At GluA2(Q) expressed in HEK293 cells (calcium flux experiment), the most potent compound was 11m (4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, EC50=2.0 nM). The Hill coefficient in the screening and the shape of the dimerization curve in small angle X-ray scattering (SAXS) experiments using isolated GluA2 ligand-binding domain (GluA2-LBD) is consistent with binding of one molecule of 11m per dimer interface, contrary to most benzothiadiazine dioxides developed to date. This observation was confirmed by the X-ray structure of 11m bound to GluA2-LBD and by NMR. This is the first benzothiadiazine dioxide AMPApam to reach the nanomolar range.

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Section: Methodsmentioning

confidence: 99%

7-Phenoxy-Substituted 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides as Positive Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors with Nanomolar Potency

et al. 2017

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“…AMBIMETER (Svergun, DI, 1992) and GNOM (Semenyuk, AV, et al, 1991) were used to initially assess the quality of the scattering data and particle homogeneity. RAW (Hopkins, JB, et al, 2017) and PRIMUS (Konarev, PV, et al, 2003) were used for processing of the SEC-SAXS data and to generate Kratky plots. Further analysis including dimensionless Kratky were carried out with ScÅtter (Rambo, RP, et al, 2013).…”

Section: Size-exclusion Chromatography Small-angle X-ray Scattering (mentioning

confidence: 99%

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Muok

Chua

Srivastava

et al. 2020

Preprint

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Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryoelectron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a "dipped conformation" that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

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“…SAXS data were recorded on a Pilatus 100 K-S detector at 2 s per frame with a fixed camera length of 1.522 m and an energy of 9.91 keV, allowing the collection of the angular range q of 0.01-0.30 Å -1 . Primary reduction of the SAXS data was performed using RAW [51]. A Guinier plot of the buffer-subtracted profile was linear to the lowest measured q value.…”

Section: Small-angle X-ray Scatteringmentioning

confidence: 99%

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme)

Hashimoto

Kafková

Raczkowski

et al. 2019

Preprint

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247 words Text: 5581 words 6 Figures and 3 tables Figure legends: 752 words Graphical Abstract Highlights • The crystal structure of Trypanosoma brucei protein arginine methyltransferase 1 (PRMT1) is reported • Two enzyme-prozyme heterodimers form a stable heterotetramer essential for catalysis • The catalytically dead prozyme lacks elements essential for AdoMet binding • The enzyme alone is unstable and cannot form a canonical dimer due to steric clashes • T. brucei PRMT1 prozyme adopts a chaperone function conserved across kinetoplastids Enzyme Enzyme Prozyme Prozyme AdoHcy η2 η2 AdoHcy Abstract Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex.Here we present the x-ray crystal structure of the ENZ-Δ52PRO tetrameric complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.4 Å resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of a non-conserved tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRTMs in that it lacks the conserved η2 310-helix within the Rossmann fold, abolishing co-factor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding.Heterotetramerization is required for catalysis, since heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity towards the substrate protein TbRGG1. Together, we provide a structural basis for PRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the PRMT1 prozyme, which represents a novel mode of PRMT regulation. arranged at the vertices of a rhomboid structure in the most predominant 2D class average (Fig. 2b). The complex measures about 140 Å in its longest dimension, which fits the maximum distance of 143 Å obtained in a pair-distance distribution by SEC-SAXS ( Fig. 2a). Finally, a stoichiometry of 1:1 was obtained for the TbPRMT1 heterotetramer with a Maltose Binding Protein (MBP)-fused TbRGG1 substrate by isothermal titration calorimetry (ITC) ( Fig. 2c and Table 1) [14]. Collectively, these data demonstrate that the TbPRMT1 complex forms a rigid heterotetrameric unit in solution. The TbPRMT1 PRO N-terminus contributes to substrate recognitionPrevious studies have shown that all PRMTs contain a highly conserved core domain comprising...

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BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis

Cited by 546 publications

References 66 publications

7-Phenoxy-Substituted 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides as Positive Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors with Nanomolar Potency

7-Phenoxy-Substituted 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides as Positive Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors with Nanomolar Potency

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme)

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