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Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.
The combination of high-throughput sequencing and in vivo crosslinking approaches leads to the progressive uncovering of the complex interdependence between cellular transcriptome and proteome. Yet, the molecular determinants governing interactions in protein-RNA networks are not well understood. Here we investigated the relationship between the structure of an RNA and its ability to interact with proteins. Analysing in silico, in vitro and in vivo experiments, we find that the amount of double-stranded regions in an RNA correlates with the number of protein contacts. This relationship —which we call structure-driven protein interactivity— allows classification of RNA types, plays a role in gene regulation and could have implications for the formation of phase-separated ribonucleoprotein assemblies. We validate our hypothesis by showing that a highly structured RNA can rearrange the composition of a protein aggregate. We report that the tendency of proteins to phase-separate is reduced by interactions with specific RNAs.
Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.
Multiplicity dependence of inclusive J/ψ production at midrapidity in pp collisions at √s = 13 TeV
(2020)
Measurements of the inclusive J/ψ yield as a function of charged-particle pseudorapidity density dNch/dη in pp collisions at √s = 13 TeV with ALICE at the LHC are reported. The J/ψ meson yield is measured at midrapidity (|y| < 0.9) in the dielectron channel, for events selected based on the charged-particle multiplicity at midrapidity (|η| < 1) and at forward rapidity (−3.7 < η < −1.7 and 2.8 < η < 5.1); both observables are normalized to their corresponding averages in minimum bias events. The increase of the normalized J/ψ yield with normalized dNch/dη is significantly stronger than linear and dependent on the transverse momentum. The data are compared to theoretical predictions, which describe the observed trends well, albeit not always quantitatively.
Z-boson production in p-Pb collisions at √sNN = 8.16 TeV and Pb-Pb collisions at √sNN = 5.02 TeV
(2020)
Measurement of Z-boson production in p-Pb collisions at sNN−−−√ = 8.16 TeV and Pb-Pb collisions at sNN−−−√ = 5.02 TeV is reported. It is performed in the dimuon decay channel, through the detection of muons with pseudorapidity −4 < ημ < −2.5 and transverse momentum pμT > 20 GeV/c in the laboratory frame. The invariant yield and nuclear modification factor are measured for opposite-sign dimuons with invariant mass 60 < mμμ < 120 GeV/c2 and rapidity 2.5 < yμμcms < 4. They are presented as a function of rapidity and, for the Pb-Pb collisions, of centrality as well. The results are compared with theoretical calculations, both with and without nuclear modifications to the Parton Distribution Functions (PDFs). In p-Pb collisions the center-of-mass frame is boosted with respect to the laboratory frame, and the measurements cover the backward (−4.46 < yμμcms < −2.96) and forward (2.03 < yμμcms < 3.53) rapidity regions. For the p-Pb collisions, the results are consistent within experimental and theoretical uncertainties with calculations that include both free-nucleon and nuclear-modified PDFs. For the Pb-Pb collisions, a 3.4σ deviation is seen in the integrated yield between the data and calculations based on the free-nucleon PDFs, while good agreement is found once nuclear modifications are considered.
The inclusive J/ψ elliptic (v2) and triangular (v3) flow coefficients measured at forward rapidity (2.5 < y < 4) and the v2 measured at midrapidity (|y| < 0.9) in Pb-Pb collisions at sNN−−−√ = 5.02 TeV using the ALICE detector at the LHC are reported. The entire Pb-Pb data sample collected during Run 2 is employed, amounting to an integrated luminosity of 750 μb−1 at forward rapidity and 93 μb−1 at midrapidity. The results are obtained using the scalar product method and are reported as a function of transverse momentum pT and collision centrality. At midrapidity, the J/ψ v2 is in agreement with the forward rapidity measurement. The centrality averaged results indicate a positive J/ψ v3 with a significance of more than 5σ at forward rapidity in the pT range 2 < pT < 5 GeV/c. The forward rapidity v2, v3, and v3/v2 results at low and intermediate pT (pT ≲ 8 GeV/c) exhibit a mass hierarchy when compared to pions and D mesons, while converging into a species-independent curve at higher pT. At low and intermediate pT, the results could be interpreted in terms of a later thermalization of charm quarks compared to light quarks, while at high pT, path-length dependent effects seem to dominate. The J/ψ v2 measurements are further compared to a microscopic transport model calculation. Using a simplified extension of the quark scaling approach involving both light and charm quark flow components, it is shown that the D-meson vn measurements can be described based on those for charged pions and J/ψ flow.
The invariant differential cross section of inclusive ω(782) meson production at midrapidity (|y|<0.5) in pp collisions at s√=7TeV was measured with the ALICE detector at the LHC over a transverse momentum range of 2<pT<17GeV/c. The ω meson was reconstructed via its ω→π+π−π0 decay channel. The measured ω production cross section is compared to various calculations: PYTHIA 8.2 Monash 2013 describes the data, while PYTHIA 8.2 Tune 4C overestimates the data by about 50%. A recent NLO calculation, which includes a model describing the fragmentation of the whole vector-meson nonet, describes the data within uncertainties below 6GeV/c, while it overestimates the data by up to 50% for higher pT. The ω/π0 ratio is in agreement with previous measurements at lower collision energies and the PYTHIA calculations. In addition, the measurement is compatible with transverse mass scaling within the measured pT range and the ratio is constant with Cω/π0=0.67±0.03~(stat)~±0.04~(sys)~ above a transverse momentum of 2.5GeV/c.
Systematic studies of charge-dependent two- and three-particle correlations in Pb-Pb collisions at sNN−−−√ = 2.76 and 5.02 TeV used to probe the Chiral Magnetic Effect (CME) are presented. These measurements are performed for charged particles in the pseudorapidity (η) and transverse momentum (pT) ranges |η| < 0.8 and 0.2 < pT < 5 GeV/c. A significant charge-dependent signal that becomes more pronounced for peripheral collisions is reported for the CME-sensitive correlators γ1, 1 = 〈cos(φα + φβ − 2Ψ2)〉 and γ1, − 3 = 〈cos(φα − 3φβ + 2Ψ2)〉. The results are used to estimate the contribution of background effects, associated with local charge conservation coupled to anisotropic flow modulations, to measurements of the CME. A blast-wave parametrisation that incorporates local charge conservation tuned to reproduce the centrality dependent background effects is not able to fully describe the measured γ1,1. Finally, the charge and centrality dependence of mixed-harmonics three-particle correlations, of the form γ1, 2 = 〈cos(φα + 2φβ − 3Ψ3)〉, which are insensitive to the CME signal, verify again that background contributions dominate the measurement of γ1,1.
The elliptic and triangular flow coefficients v2 and v3 of prompt D0, D+, and D∗+ mesons were measured at midrapidity (|y| < 0.8) in Pb–Pb collisions at the centre-of-mass energy per nucleon pair of √sNN = 5.02 TeV with the ALICE detector at the LHC. The D mesons were reconstructed via their hadronic decays in the transverse momentum interval 1 < pT < 36 GeV/c in central (0–10%) and semi-central (30–50%) collisions. Compared to pions, protons, and J/ψ mesons, the average D-meson vn harmonics are compatible within uncertainties with a mass hierarchy for pT 3 GeV/c, and are similar to those of charged pions for higher pT. The coupling of the charm quark to the light quarks in the underlying medium is further investigated with the application of the event-shape engineering (ESE) technique to the D-meson v2 and pT-differential yields. The D-meson v2 is correlated with average bulk elliptic flow in both central and semi-central collisions. Within the current precision, the ratios of per-event Dmeson yields in the ESE-selected and unbiased samples are found to be compatible with unity. All the measurements are found to be reasonably well described by theoretical calculations including the effects of charm-quark transport and the recombination of charm quarks with light quarks in a hydrodynamically expanding medium.
Cryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.