Resolution of<i>ab initio</i>shapes determined from small-angle scattering

Tuukkanen, Anne; Kleywegt, Gerard J.; Svergun, Dmitri I.

doi:10.1107/s2052252516016018

Cited by 88 publications

(65 citation statements)

References 35 publications

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“…To this end, we used EOM to investigate the different conformations of AfSBDS. The protein was divided into its three domains according to the crystallographic structure; the inter-domain linkers were removed, and domain 2 was fixed, while A low-resolution model of the protein can be reconstructed from the SAXS data using ab-initio algorithms such as DAMMIN [38], DAMMIF [39], MONSA [38], or by hybrid modeling using known atomic structures of domains with SASRES [40]. The ab-initio methods represent the particle as finite volume elements (or dummy atoms) and employ a simulated annealing to fit the experimental data.…”

Section: Resultsmentioning

confidence: 99%

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

et al. 2018

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Abstract:The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS) has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome) protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N-and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

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Section: Resultsmentioning

confidence: 99%

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

et al. 2018

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“…BioSAXS techniques are applied to analyse tertiary and quaternary structures or even time-resolved folding, degradation or complex formation of (biological) macromolecules in solution, utilizing X-ray scattering intensity patterns at small angles (Franke et al, 2012;Graceffa et al, 2013;Sviridova et al, 2017). Applying ab initio modelling techniques the shape of macromolecules can be calculated (Tuukkanen et al, 2016;Franke et al, 2017). At the BioSAXS beamline P12, data collection can be performed in batch or in flow mode.…”

Section: Introductionmentioning

confidence: 99%

Multi-channelin situdynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12

Dierks¹,

Blanchet

Graewert

et al. 2018

J Synchrotron Radiat

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Small-angle X-ray scattering (SAXS) analysis of biomolecules is increasingly common with a constantly high demand for comprehensive and efficient sample quality control prior to SAXS experiments. As monodisperse sample suspensions are desirable for SAXS experiments, latest dynamic light scattering (DLS) techniques are most suited to obtain non-invasive and rapid information about the particle size distribution of molecules in solution. A multi-receiver four-channel DLS system was designed and adapted at the BioSAXS endstation of the EMBL beamline P12 at PETRA III (DESY, Hamburg, Germany). The system allows the collection of DLS data within round-shaped sample capillaries used at beamline P12. Data obtained provide information about the hydrodynamic radius of biological particles in solution and dispersity of the solution. DLS data can be collected directly prior to and during an X-ray exposure. To match the short X-ray exposure times of around 1 s for 20 exposures at P12, the DLS data collection periods that have been used up to now of 20 s or commonly more were substantially reduced, using a novel multi-channel approach collecting DLS data sets in the SAXS sample capillary at four different neighbouring sample volume positions in parallel. The setup allows online scoring of sample solutions applied for SAXS experiments, supports SAXS data evaluation and for example indicates local inhomogeneities in a sample solution in a time-efficient manner. Biological macromolecules with different molecular weights were applied to test the system and obtain information about the performance. All measured hydrodynamic radii are in good agreement with DLS results obtained by employing a standard cuvette instrument. Moreover, applying the new multi-channel DLS setup, a reliable radius determination of sample solutions in flow, at flow rates normally used for size-exclusion chromatography-SAXS experiments, and at higher flow rates, was verified as well. This study also shows and confirms that the newly designed sample compartment with attached DLS instrumentation does not disturb SAXS measurements.

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“…Additionally, the MW estimates were derived from the scattering data based on the hydrated volume Vp computed using Porod analysis . Resolutions of the ab initio model ensembles were computed using a Fourier shell correlation (FSC)‐based approach (Appendix Table S1) .…”

Section: Methodsmentioning

confidence: 99%

cGAS facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity

et al. 2019

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Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING‐dependent release of type I IFN. Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe‐ and danger‐associated molecular patterns that contribute to host–microbe crosstalk during health and disease.

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Resolution ofab initioshapes determined from small-angle scattering

Cited by 88 publications

References 35 publications

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

Multi-channelin situdynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12

cGAS facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity

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