Direct observation of molecular arrays in the organized smooth endoplasmic reticulum

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization. Results Here, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation - do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets. Conclusion Our results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering.Published versio

Structural basis of sterol recognition by human hedgehog receptor PTCH1

Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4-Å cryo-EM structure of the human PTCH1 bound to ShhNC24II, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by 10 sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol-sensing domain (SSD) and the SSD-like domain (SSDL), which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.ISSN:2375-254

A Meta-Brokering Framework for Science Gateways

Recently scientific communities produce a growing number of computation-intensive applications, which calls for the interoperation of distributed infrastructures including Clouds, Grids and private clusters. The European SHIWA and ER-flow projects have enabled the combination of heterogeneous scientific workflows, and their execution in a large-scale system consisting of multiple Distributed Computing Infrastructures. One of the resource management challenges of these projects is called parameter study job scheduling. A parameter study job of a workflow generally has a large number of input files to be consumed by independent job instances. In this paper we propose a meta-brokering framework for science gateways to support the execution of such workflows. In order to cope with the high uncertainty and unpredictable load of the utilized distributed infrastructures, we introduce the so called resource priority services. These tools are capable of determining and dynamically updating priorities of the available infrastructures to be selected for job instances. Our evaluations show that this approach implies an efficient distribution of job instances among the available computing resources resulting in shorter makespan for parameter study workflows

Structure of the connexin-43 gap junction channel in a putative closed state

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state

Structural basis of adenylyl cyclase 9 activation

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation

Structural basis of adenylyl cyclase 9 activation

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation

Digital system of registering, storage and actualization of municipal legal acts: elaboration and approbation in the territory of Krasnoyarsk krai

Objective to determine the legal status of the electronic information system of accounting storing and updating of municipal normative legal acts to disseminate the experience of development and implementation of such a system in Krasnoyarsk krai. Methods general scientific method of dialectical cognition historicallegal systemstructural comparativelegal formallogical and other particular scientific methods of cognition. Results the authors come to the conclusion that the electronic information system of accounting storing and updating of municipal normative legal acts is a type of official information systems and has special qualitative characteristics. Development and implementation of such systems in the activities of local governments according to the authors is a promising direction to ensure the openness of municipal legal acts and the accessibility of information on the results of municipal rulemaking. To illustrate the authorrsquos approach a functional description of the electronic information system of accounting storing and updating of municipal regulatory legal acts ldquoMunicipal legal acts of the Krasnoyarsk krairdquo is given. Scientific novelty consists in the development of theoretical conceptions about the legal nature of official information systems and applied problems of their use in the activities of local governments. Modern information and communication technologies allow the transition to the real embodiment of direct democracy including the involvement of the population in the direct decisionmaking at the level of local government using EISof municipal legal acts. Given the functions that may be performed by EIS ensuring access of citizens to the system of legal acts their storage and systematization communication between local governments and public authorities provision of official authentic texts of a legislative act organization of discussion on drafts of municipal legal acts formation of open data etc. it seems necessary to legislatively establish a legal regime of EIS as the official legal information system. EIS has significant advantages in comparison with other legal systems including the register maintained by the Ministry of Justice of the Russian Federation on the website ldquoNormative legal acts of the Russian Federationrdquo. Additional features of EIS aimed at the implementation of the provisions of the Federal Law ldquoOn ensuring access to information on the activities of state bodies and local selfgovernmentrdquo may occur when the legislator makes changes to the procedure for the official publication of municipal legal acts provided for in part 2 of Article 47 of the Federal Law ldquoOn general principles of local selfgovernment in the Russian Federationrdquo. Practical significance consist in the possibility of wide implementation of the electronic information system of accounting storing and updating of municipal normative legal acts in the activities of local governments developed by the authorsrsquo team