The ER morphology-regulating lunapark protein induces the formation of stacked bilayer discs

This is the final version of the article. Available from the publisher via the DOI in this recordLunapark (Lnp) is a conserved membrane protein that localizes to and stabilizes three-way junctions of the tubular ER network. In higher eukaryotes, phosphorylation of Lnp may contribute to the conversion of the ER from tubules to sheets during mitosis. Here, we report on the reconstitution of purified Lnp with phospholipids. Surprisingly, Lnp induces the formation of stacked membrane discs. Each disc is a bicelle, with Lnp sitting in the bilayer facing both directions. The interaction between bicelles is mediated by the cytosolic domains of Lnp, resulting in a constant distance between the discs. A phosphomimetic Lnp mutant shows reduced bicelle stacking. Based on these results, we propose that Lnp tethers ER membranes in vivo in a cell cycle–dependent manner. Lnp appears to be the first membrane protein that induces the formation of stacked bicelles.S Wang was supported by a fellowship from the Charles King Trust and RE Powers by a NIGMS T32 training grant (GM008313). We acknowledge the Max Planck Society and University of Exeter for supporting V Gold, in particular Werner Kühlbrandt and Deryck Mills at the Max Planck Institute of Biophysics. TA Rapoport is a Howard Hughes Medical Institute Investigator

Structure of a type IV pilus machinery in the open and closed state.

This is the final version of the article. Available from eLife Sciences Publications via the DOI in this record.Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ~30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (AV 9/6-1)

Visualizing active membrane protein complexes by electron cryotomography.

This is the final version of the article. Available from Nature Publishing Group via the DOI in this record.Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.We thank Drs Ulrike Endesfelder and Mike Heilemann (Institute of Physical and Theoretical Chemistry, University of Frankfurt) for help with confocal microscopy, Deryck Mills (MPI of Biophysics, Frankfurt) for maintenance of the EM facility, and Paolo Lastrico (Graphics Department, MPI of Biophysics, Frankfurt) for assistance with Supplementary Movies and Fig. 1a. We thank Drs Bertram Daum and Karen Davies for helpful discussions on tomography. The plasmids pMAL-c2x-MT2 and pMAL-c2x-MT3 were a gift from Dr Christina Risco (CNB-CSIC, Madrid). This work was supported by the Max Planck Society, Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 746), Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS) and by an EMBO Long-Term Fellowship to V.A.M.G. (ALTF 1035-2010)

Topology and structure/function correlation of ring- and gate-forming domains in the dynamic secretin complex of Thermus thermophilus.

This is the final version of the article. Available from the publisher via the DOI in this record.Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural transformation of DNA in the thermophilic bacterium Thermus thermophilus requires a unique secretin complex comprising six stacked rings, a membrane-embedded cone structure, and two gates that open and close a central channel. To investigate the role of distinct domains in ring and gate formation, we examined a set of deletion derivatives by cryomicroscopy techniques. Here we report that maintaining the N0 ring in the deletion derivatives led to stable PilQ complexes. Analyses of the variants unraveled that an N-terminal domain comprising a unique βββαβ fold is essential for the formation of gate 2. Furthermore, we identified four βαββα domains essential for the formation of the N2 to N5 rings. Mutant studies revealed that deletion of individual ring domains significantly reduces piliation. The N1, N2, N4, and N5 deletion mutants were significantly impaired in T4P-mediated twitching motility, whereas the motility of the N3 mutant was comparable with that of wild-type cells. This indicates that the deletion of the N3 ring leads to increased pilus dynamics, thereby compensating for the reduced number of pili of the N3 mutant. All mutants exhibit a wild-type natural transformation phenotype, leading to the conclusion that DNA uptake is independent of functional T4P.This work was supported by Deutsche Forschungsgemeinschaft Grant AV 9/6-1. The authors declare that they have no conflicts of interest with the contents of this article

Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography

This is the final version of the article. Available from the publisher via the DOI in this recordThe video component of this article can be found at http://www.jove.com/video/51228/Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.This work was supported by the Max Planck Society (K.M.D., B.D., A.W.M., T.B., T.B.B., D.J.M., and W.K.), the Cluster of Excellence Frankfurt “Macromolecular Complexes” funded by the Deutsche Forschungsgemeinschaft (W.K.) and a postdoctoral EMBO Long-Term Fellowship (V.A.M.G.)

Architecture and modular assembly of Sulfolobus S-layers revealed by electron cryotomography

This is the final version. Available from the National Academy of Sciences of the United States of America via the DOI in this record. Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.ER

Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium

This is the final version. Available on open access from Nature Research via the DOI in this recordData availability: EM maps have been deposited in the Electron Microscopy Data Bank (EMDB, https://www.ebi.ac.uk/pdbe/emdb/) with accession codes EMD-10647 (wide pilus, PilA4) and EMD-10648 (narrow pilus, PilA5). Models have been deposited in the Protein Data Bank (PDB, https://www.rcsb.org/) with accession codes 6XXD (PilA4) and 6XXE (PilA5). The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE75 partner repository (https://www.ebi.ac.uk/pride/) with dataset identifier PXD017353. The source data underlying Figs. 5c–e and 6, Supplementary Figs. 2, 3b, c, 4a–c, 5e and 6e, f, and Supplementary Table 4 are provided in the Source Data file. Uncropped versions of gels and blots (for Fig. 5c, d, and Supplementary Figs. 2b and 4a–c) and twitching images (for Fig. 6a) are also shown in Supplementary Fig. 10.Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus (‘wide’ and ‘narrow’), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.Biotechnology & Biological Sciences Research Council (BBSRC)Max-Planck-SocietyUniversity of ExeterDeutsche Forschungsgemeinschaf

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage

This is the final version. Available on open access from Nature Research via the DOI in this record. The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.Wellcome TrustBiotechnology and Biological Sciences Research Council (BBSRC)Medical Research Council (MRC)Gordon and Betty Moore FoundationEuropean Research Council (ERC)Biotechnology and Biological Sciences Research CouncilAustralian Postgraduate Award (APA)IMB Research Advancement Awar

Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius

This is the author accepted manuscript. The final version is available from eLife Sciences Publications via the DOI in this recordData availability: The atomic coordinates of SlaA were deposited in the Protein Data Bank (https://www.rcsb.org/) with accession numbers PDB-7ZCX, PDDB-8AN3, and PDB-8AN3 for pH 4, 7 and 10, respectively. The electron density maps were deposited in the EM DataResource (https://www.emdataresource.org/) with accession numbers EMD-14635, EMD-15531 and EMD-15531 for pH 4, 7 and 10, respectively. Sub-tomogram averaging map of the S-layer has been deposited in the EMDB (EMD-18127) and models of the hexameric and trimeric pores in the Protein Databank under accession codes PDB-8QP0 and PDB-8QOX, respectivelyOther structural data used in this study are: H. volcanii csg (PDB ID: 7PTR, http://dx.doi.org/10.2210/pdb7ptr/pdb), and C. crescentus RsaA ((N-terminus PDB ID: 6T72, http://dx.doi.org/10.2210/pdb6t72/pdb, C-terminus PDB ID: 5N8P, http://dx.doi.org/10.2210/pdb5n8p/pdb).Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single particle cryo electron microscopy (cryoEM), cryo electron tomography (cryoET) and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesise that jackknife-like conformational changes, changes play important roles in S-layer assembly.European Research CouncilWellcome TrustWellcome TrustAgence Nationale de la RechercheAgence Nationale de la RechercheLeverhulme TrustBiotechnology and Biological Sciences Research Council (BBSRC