We here present a structural model for the cyclic nucleotide-modulated potassium channel homolog from Mesorhizobium loti, MloK1, determined from 2D crystals in the presence of lipids. The structure provides a clear picture of the arrangement of the cyclic nucleotide-binding domains with respect to both the pore and the putative voltage sensor domains when cAMP is bound, and reveals a potential gating mechanism in the context of the lipid-embedded channel.
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All four compounds identified by unit cell parameters using MicroED data from within the same grid square. Credit: ChemRxiv. More information: Tim Gruene et al. Rapid structure determination of microcrystalline molecular compounds using electron diffraction, Angewandte Chemie International Edition DOI: This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no part may be reproduced without the written permission. The content is provided for information purposes only. Daily rainfall over Sumatra linked to larger atmospheric phenomenon Sep 20, Nevertheless, the way the two rings interact in the dodecamer and how this results in different conformations is still not known in molecular detail.
The four crystal structures are very similar with r. The orthologous Rvb1 and Rvb2 genes were identified due to their high sequence conservation across the eukaryotic kingdom. We separately expressed Rvb1 and Rvb2 in E. The Rvb2 profile shows a small peak that might indicate the presence of oligomers in the sample. However, negative stain electron microscopy EM grids of purified Rvb2 showed that it is monomeric under these conditions S2A Fig. Molecular weight standards corresponding to , , 44 and 17 kDa are shown on top. In this arrangement the hetero-hexameric rings are rotated with respect to each other by 60 degree, hence Rvb1 and Rvb2 are stacked on top of each other.
Rvb1 and Rvb2 proteins are depicted in gold and blue, respectively.
Structural Electron Crystallography
The relative orientation of the rings is illustrated by the cartoons. The position of the DII domain in Rvb2 agrees with the prediction based on molecular dynamics simulations [ 17 ]. Hence, the described interface requires two distinct conformations of the DII domains from Rvb1 and Rvb2 to form the dodecameric complex.
The SeMet crystals revealed density in Rvb1 that could be identified as ADP, whereas Rvb2 clearly shows two density peaks at the same position most likely corresponding to sulphates Fig 4A present in the crystallization solution and known to mimic phosphates. In the crystal structure of the native protein solved using the SeMet map as a starting model we observed very similar densities in Rvb1 and Rvb2 that could be identified as ADP molecules.
In contrast, we observe subtle changes in the case of Rvb2. No apparent differences between the two nucleotide-binding states were found in the dodecameric assemblies. This is further supported by maximum likelihood superimpositions and B-Factor calculations [ 22 , 23 ] that highlight the flexibility around the Rvb2 nucleotide pocket Fig 4D. The heat map also indicates that in both nucleotide states the linkers in Rvb1 and Rvb2 show the highest mobility in the middle part residues — and to in Rvb1; and — in Rvb2.
Interestingly, this region is connected through a well-structured N-terminal tail with the catalytic sites in Rvb1 and Rvb2. We speculate that the N-terminal tail may relay conformational changes between active sites and the DII domains, which were proposed to be flexible by molecular dynamics simulations [ 5 , 17 ].
Rvb1 is shown in the left and Rvb2 in the right, catalytic motifs are colour coded as stated below a. The experimental density of the nucleotides is depicted as mesh.businesspodden.se/cuando-te-grita-el-alma-la-gua.php
MS Electron Crystallography as a Tool for Structure Solution and Refinement
The micrograph field in Fig 5A shows typical top and side views. None of the reference-free averaged images Fig 5B showed the core-core conformation, as indicated by comparison with the projections of the electron density of the crystal structure Fig 5C and by the presence of three layers of density that have been previously described. Accordingly, the two outer electron dense layers correspond to the hetero-hexameric rings in side views, while the middle region comprises the flexible DII domains. Furthermore, the outer layers show high resolution features, while the middle region remains more blurry.
However, the abundance of the compact conformation observed in Chaetomium thermophilum is in agreement with previous studies in yeast and human [ 7 , 13 , 16 ].
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Unevenly distributed densities can be also distinguished in the periphery of the top view-rings. This asymmetric arrangement appears to contradict the D3 symmetry observed in the crystal structure, suggesting that there are variations in the interaction between the rings in solution. The comparison of the compact 2D averages with the projections of the crystal structure confirms this observation, as none of the projections showed slanted rings or elongated channels Fig 5C. On the other hand, all the stretched 2D averages showed straight rings and match in height with the projections of the electron density of the crystal structure, although the middle part is slightly different, suggesting a flexible DII position.
The fact that the averaged side views show slanted and straight rings raised the question whether the asymmetric features reflects additional conformational states or several views of the same complex. We therefore performed a three dimensional 3D reconstruction to elucidate how the asymmetry of the complex relates to the different conformational states. Complexes in side and top views are highlighted. Scale bar, 50 nm. Top views are represented in the two upper rows and side views in the two lower rows. The stretched classes are marked with an asterisk.
The images corresponding to the stretched and compact conformations were split based on the 2D classification, and each dataset was processed independently.
Straight and slanted rings are present in the re-projections of the compact 3D model confirming that the different degrees of displacement present in the 2D averages could in principle be explained by a single 3D conformation—at least at the given resolution. Although the homogeneity of dodecamers in our purification and the abundance of compact conformation justified the use of top and side views for the reconstruction, we also generate a 3D model only from side views, as previously described [ 7 , 13 ].
It is thus obvious to conclude that the rings would correspond to the core domains while the flexible DII domains localize in the middle part. The slanted position of the rings becomes apparent when rotating the complex along the longitudinal axis S6C Fig , and is also evident in the top view, where parts of the bottom ring are also visible Fig 6A. However, further information is needed regarding the biological function of the slanted rings.
The tilted arrangement of two hexameric rings has been observed in other helicases such us the MCM [ 28 ] and the SV40 large tumor antigen [ 29 ], both suggesting the implication of the ring offset in the initiation of DNA replication. We currently favour the idea that the plasticity of the rings within the dodecamer might facilitate the asymmetric interaction with other proteins e.
INO80 complex. Stretched side views were further classified in 3D and particles were used to obtain a D3-symmetrised volume Fig 6B at Although the 3D models were refined independently with and without symmetry, the re-projections of the D3 symmetrised model correlate better with the stretched reference-free 2D class averages Fig 6B and S7B Fig.
Given the reduced number of particles present in the stretched 3D model, the better correlation may partly be due to imposing additional rotational symmetry. Thus, the stretched models with and without D3 symmetry imposition were compared S7C Fig and confirmed that applying D3 symmetry was indeed justified. Hence, the core-core arrangement appears not to be present in solution.
The final resolution of the stretched complex is in accordance with the features shown by the 2D class averages and the limited number of particles in the final model. In contrast, the high resolution features apparent in some of the 2D class averages are missing in the final compact model Figs 5B and 6A. A recent study in yeast also pointed out that the presence of multiple intermediates in low amounts prevented a high resolution structure determination [ 13 ].
We can thus speculate that the presence of conformational transitions, between the stretched and compact conformations, and specially the flexibility of the DII domains may have prevented the alignment to converge to a high resolution structure. This became evident during the 2D classification, where only some of the averaged images showed high resolution features and from those just a fraction had a well-defined DII interface thereby considerably reducing the number of similar particles contributing to the final model.