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Abstract: Legionella pneumophila is a bacterial pathogen that utilises a Type IV secretion (T4S) system to inject effector proteins into human macrophages. Essential to the recruitment and delivery of effectors to the T4S machinery is the membrane-embedded T4 coupling complex (T4CC). Here, we purify an intact T4CC from the Legionella membrane. It contains the DotL ATPase, the DotM and DotN proteins, the chaperone module IcmSW, and two previously uncharacterised proteins, DotY and DotZ. The atomic resolution structure reveals a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module protrudes. Six of these hetero-pentameric complexes may assemble into a 1.6-MDa hexameric nanomachine, forming an inner membrane channel for effectors to pass through. Analysis of multiple cryo EM maps, further modelling and mutagenesis provide working models for the mechanism for binding and delivery of two essential classes of Legionella effectors, depending on IcmSW or DotM, respectively.

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Abstract: We measure the electronic structure of FeSe from within individual orthorhombic domains. Enabled by an angle-resolved photoemission spectroscopy beamline with a highly focused beam spot (nano-ARPES), we identify clear stripelike orthorhombic domains in FeSe with a length scale of approximately 1–5 μm. Our photoemission measurements of the Fermi surface and band structure within individual domains reveal a single electron pocket at the Brillouin zone corner. This result provides clear evidence for a one-electron-pocket electronic structure of FeSe, observed without the application of uniaxial strain, and calls for further theoretical insight into this unusual Fermi surface topology. Our results also showcase the potential of nano-ARPES for the study of correlated materials with local domain structures.

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Abstract: The crystal structure of the organic pigment 2-mono­methyl-quinacridone (Pigment Red 192, C21H14N2O2) was solved from X-ray powder diffraction data. The resulting average structure is described in space group [P\overline 1], Z = 1 with the molecule on the inversion centre. The molecules are arranged in chains. The molecules, which have no inversion symmetry, show orientational head-to-tail disorder. In the average structure, the methyl group is disordered and found on both ends of the molecule with an occupancy of 0.5 each. The disorder and the local structure were investigated using various ordered structural models. All models were analysed by three approaches: Rietveld refinement, structure refinement to the pair distribution function (PDF) and lattice-energy minimization. All refinements converged well. The Rietveld refinement provided the average structure and gave no indication of a long-range ordering. The refinement to the PDF turned out to be very sensitive to small structural details, giving insight into the local structure. The lattice-energy minimizations revealed a significantly preferred local ordering of neighbouring molecules along the [0[\bar 1]1] direction. In conclusion, all methods indicate a statistical orientational disorder with a preferred parallel orientation of molecules in one direction. Additionally, electron diffraction revealed twinning and faint diffuse scattering.

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Abstract: Background: Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. Methods: Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. Results: The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

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Abstract: Coordination cages are well-known to act as molecular containers that can bind small-molecule guests in their cavity. Such cavity binding is associated with interactions of the guests with the surrounding set of surfaces that define the cavity; a guest that is a good fit for the cavity will have many favourable interactions with the interior surfaces of the host. As cages have exterior as well as interior surfaces, possibilities also exist for ‘guests’ that are not well-bound in the cavity to interact with the exterior surface of the cage where spatial constraints are fewer. In this paper, we report a combined solid-state and solution study using an octanuclear cubic M8L12 coordination cage which illustrates the occurrence of both types of interaction. Firstly, crystallographic studies show that a range of guests bind inside the cavity (either singly or in stacked pairs) and/or interact with the cage exterior surface, depending on their size. Secondly, fluorescence titrations in aqueous solution show how some flexible aromatic disulfides show two separate types of interaction with the cage, having different spectroscopic consequences; we ascribe this to separate interactions with the exterior surface and the interior surface of the host cage with the former having a higher binding constant. Overall, it is clear that the idea of host/guest interactions in molecular containers needs to take more account of external surface interactions as well as the obvious cavity-based binding.