Advances in automated data analysis and processing within <i>autoPROC</i>, combined with improved characterisation, mitigation and visualisation of the anisotropy of diffraction limits using <i>STARANISO</i>

Vonrhein, Clemens; Tickle, Ian J.; Flensburg, Claus; Keller, Philip; Paciorek, W. A.; Sharff, A.; Bricogne, G.

doi:10.1107/s010876731809640x

Cited by 118 publications

(97 citation statements)

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“…Data integration and scaling were performed using the autoPROC implementation of XDS and AIMLESS ( Vonrhein et al, 2011 ). Anisotropy correction of the eVP40 P6 4 22 data set was performed using STARANISO with a surface threshold of 1.2/σ(I), implemented through the autoPROC pipeline ( Vonrhein et al, 2018 ). Isotropic data were used for model building and refinement of the eVP40 P6 2 crystal form and anisotropic corrected data were used for model building and refinement of the eVP40 P6 4 22 crystal form.…”

Section: Methodsmentioning

confidence: 99%

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers

Wan

Clarke

Norris

et al. 2020

eLife

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Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles.

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Section: Methodsmentioning

confidence: 99%

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers

Wan

Clarke

Norris

et al. 2020

eLife

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“…Aimless was then used to merge the observations into average densities for two datasets (47) to maximize the total number of observed reflections, ( Table 2). STARANISO was used as, similarly to AncFMO2, these crystals suffered severely from anisotropy (48,49). Using STARANISO on the final merged dataset was imperative for elucidating a good electron density map.…”

Section: Crystallization and Structural Determination Of Ancfmo1mentioning

confidence: 99%

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

Bailleul

Nicoll

Mascotti

et al. 2021

Journal of Biological Chemistry

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Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2’-phosphate being pushed inside the NADP + binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

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“…Crystals were cryocooled with the addition of 12% PEG400 in the mother liquor prior to X-ray diffraction, and data was collected at the Diamond Light Source (UK) at beamline i04-1. Data was indexed and integrated using the autoPROC STARANISO pipeline, using XDS [37], scaled and merged using Aimless [38] and STARANISO [39] for anisotropic correction (ellipsoidal with resolution cutoff criteria -local (I/σI) = 1.2). Molecular replacement was performed with Phaser [40] using BVMO AFL838 as search model (PDB ID 5J7X).…”

Section: Crystallization and Structure Determination Of Bvmo Afl210mentioning

confidence: 99%

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

et al. 2020

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Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that primarily convert ketones to esters, but can also catalyze heteroatom oxidation. Several structural studies have highlighted the importance of the 'control loop' in BVMOs, which adopts different conformations during catalysis. Central to the 'control loop' is a conserved tryptophan that has been implicated in NADP(H) binding. BVMO AFL210 from Aspergillus flavus, however, contains a threonine in the equivalent position. Here, we report the structure of BVMO AFL210 in complex with NADP + in both the 'open' and 'closed' conformations. In neither conformation does Thr513 contact the NADP + . Although mutagenesis of Thr513 did not significantly alter the substrate scope, changes in peroxyflavin stability and reaction rates were observed. Mutation of this position also brought about changes in the regioand enantioselectivity of the enzyme. Moreover, lower rates of overoxidation during sulfoxidation of thioanisole were also observed.

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Advances in automated data analysis and processing within autoPROC, combined with improved characterisation, mitigation and visualisation of the anisotropy of diffraction limits using STARANISO

Cited by 118 publications

References 0 publications

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

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