bims-strubi Biomed News
on Advances in structural biology
Issue of 2021–11–14
nine papers selected by
Alessandro Grinzato, European Synchrotron Radiation Facility
  1. Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography.
  2. Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT.
  3. The ion-coupling mechanism of human excitatory amino acid transporters.
  4. A site of vulnerability at V3 crown defined by HIV-1 bNAb M4008_N1.
  5. Structure and activation mechanism of the hexameric plasma membrane H+-ATPase.
  6. Structures of the HER2-HER3-NRG1β complex reveal a dynamic dimer interface.
  7. The trRosetta server for fast and accurate protein structure prediction.
  8. High-endurance micro-engineered LaB6 nanowire electron source for high-resolution electron microscopy.
  9. Room temperature XFEL crystallography reveals asymmetry in the vicinity of the two phylloquinones in photosystem I.
  1. J Vis Exp. 2021 Oct 25.
    Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography.
    Anna Bieber, Cristina Capitanio, Florian Wilfling, Jürgen Plitzko, Philipp S Erdmann.
      Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).
    DOI:  https://doi.org/10.3791/62886
  2. Nat Commun. 2021 Nov 11. 12(1): 6508
    Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT.
    Richard W Meek, James N Blaza, Jil A Busmann, Matthew G Alteen, David J Vocadlo, Gideon J Davies.
      The O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.
    DOI:  https://doi.org/10.1038/s41467-021-26796-6
  3. EMBO J. 2021 Nov 08. e108341
    The ion-coupling mechanism of human excitatory amino acid transporters.
    Juan C Canul-Tec, Anand Kumar, Jonathan Dhenin, Reda Assal, Pierre Legrand, Martial Rey, Julia Chamot-Rooke, Nicolas Reyes.
      Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co-transport transmitter into the cytoplasm with Na+ and H+ , while counter-transporting K+ to re-initiate the transport cycle. However, the molecular mechanisms underlying ion-coupled transport remain incompletely understood. Here, we present 3D X-ray crystallographic and cryo-EM structures, as well as thermodynamic analysis of human EAAT1 in different ion bound conformations, including elusive counter-transport ion bound states. Binding energies of Na+ and H+ , and unexpectedly Ca2+ , are coupled to neurotransmitter binding. Ca2+ competes for a conserved Na+ site, suggesting a regulatory role for Ca2+ in glutamate transport at the synapse, while H+ binds to a conserved glutamate residue stabilizing substrate occlusion. The counter-transported ion binding site overlaps with that of glutamate, revealing the K+ -based mechanism to exclude the transmitter during the transport cycle and to prevent its neurotoxic release on the extracellular side.
    Keywords:  X-ray crystallography; cryo-EM; neurotransmitter transport; permeation and transport; solute carrier
    DOI:  https://doi.org/10.15252/embj.2021108341
  4. Nat Commun. 2021 Nov 09. 12(1): 6464
    A site of vulnerability at V3 crown defined by HIV-1 bNAb M4008_N1.
    Kun-Wei Chan, Christina C Luo, Hong Lu, Xueling Wu, Xiang-Peng Kong.
      Identification of vulnerable sites defined by broadly neutralizing antibodies (bNAbs) on HIV-1 envelope (Env) is crucial for vaccine design, and we present here a vulnerable site defined by bNAb M4008_N1, which neutralizes about 40% of a tier-2 virus panel. A 3.2 Å resolution cryo-EM structure of M4008_N1 in complex with BG505 DS-SOSIP reveals a large, shallow protein epitope surface centered at the V3 crown of gp120 and surrounded by key glycans. M4008_N1 interacts with gp120 primarily through its hammerhead CDR H3 to form a β-sheet interaction with the V3 crown hairpin. This makes M4008_N1 compatible with the closed conformation of the prefusion Env trimer, and thus distinct from other known V3 crown mAbs. This mode of bNAb approaching the immunogenic V3 crown in the native Env trimer suggests a strategy for immunogen design targeting this site of vulnerability.
    DOI:  https://doi.org/10.1038/s41467-021-26846-z
  5. Nat Commun. 2021 Nov 08. 12(1): 6439
    Structure and activation mechanism of the hexameric plasma membrane H+-ATPase.
    Peng Zhao, Chaoran Zhao, Dandan Chen, Caihong Yun, Huilin Li, Lin Bai.
      The S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.
    DOI:  https://doi.org/10.1038/s41467-021-26782-y
  6. Nature. 2021 Nov 10.
    Structures of the HER2-HER3-NRG1β complex reveal a dynamic dimer interface.
    Devan Diwanji, Raphael Trenker, Tarjani M Thaker, Feng Wang, David A Agard, Kliment A Verba, Natalia Jura.
      Human epidermal growth factor receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex1-3 upon binding of growth factor neuregulin-1β (NRG1β). The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. Here we isolated the NRG1β-bound near full-length HER2-HER3 dimer and, using cryo-electron microscopy, reconstructed the extracellulardomain module, revealing unexpected dynamics at the HER2-HER3 dimerization interface. We show that the dimerization arm of NRG1β-bound HER3 is unresolved because the apo HER2 monomer does not undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm-binding pocket. In a structure of the oncogenic extracellular domain mutant HER2(S310F), we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. Both HER2-HER3 and HER2(S310F)-HER3 retain the capacity to bind to the HER2-directed therapeutic antibody trastuzumab, but the mutant complex does not bind to pertuzumab. Our structure of the HER2(S310F)-HER3-NRG1β-trastuzumab Fab complex reveals that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, similar to oncogenic mutations, therapeutic agents exploit the intrinsic dynamics of the HER2-HER3 heterodimer. The unique features of a singly liganded HER2-HER3 heterodimer underscore the allosteric sensing of ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via a canonical active dimer interface.
    DOI:  https://doi.org/10.1038/s41586-021-04084-z
  7. Nat Protoc. 2021 Nov 10.
    The trRosetta server for fast and accurate protein structure prediction.
    Zongyang Du, Hong Su, Wenkai Wang, Lisha Ye, Hong Wei, Zhenling Peng, Ivan Anishchenko, David Baker, Jianyi Yang.
      The trRosetta (transform-restrained Rosetta) server is a web-based platform for fast and accurate protein structure prediction, powered by deep learning and Rosetta. With the input of a protein's amino acid sequence, a deep neural network is first used to predict the inter-residue geometries, including distance and orientations. The predicted geometries are then transformed as restraints to guide the structure prediction on the basis of direct energy minimization, which is implemented under the framework of Rosetta. The trRosetta server distinguishes itself from other similar structure prediction servers in terms of rapid and accurate de novo structure prediction. As an illustration, trRosetta was applied to two Pfam families with unknown structures, for which the predicted de novo models were estimated to have high accuracy. Nevertheless, to take advantage of homology modeling, homologous templates are used as additional inputs to the network automatically. In general, it takes ~1 h to predict the final structure for a typical protein with ~300 amino acids, using a maximum of 10 CPU cores in parallel in our cluster system. To enable large-scale structure modeling, a downloadable package of trRosetta with open-source codes is available as well. A detailed guidance for using the package is also available in this protocol. The server and the package are available at https://yanglab.nankai.edu.cn/trRosetta/ and https://yanglab.nankai.edu.cn/trRosetta/download/ , respectively.
    DOI:  https://doi.org/10.1038/s41596-021-00628-9
  8. Nat Nanotechnol. 2021 Nov 08.
    High-endurance micro-engineered LaB6 nanowire electron source for high-resolution electron microscopy.
    Han Zhang, Yu Jimbo, Akira Niwata, Akihiro Ikeda, Akira Yasuhara, Cretu Ovidiu, Koji Kimoto, Takeshi Kasaya, Hideki T Miyazaki, Naohito Tsujii, Hongxin Wang, Yasushi Yamauchi, Daisuke Fujita, Shin-Ichi Kitamura, Hironobu Manabe.
      The size tunability and chemical versatility of nanostructures enable electron sources of high brightness and temporal coherence, both of which are important characteristics for high-resolution electron microscopy1-3. Despite intensive research efforts in the field, so far, only conventional field emitters based on a bulk tungsten (W) needle have been able to yield atomic-resolution images. The absence of viable alternatives is in part caused by insufficient fabrication precision for nanostructured sources, which require an alignment precision of subdegree angular deviation of a nanometre-sized emission area with the macroscopic emitter axis4. To overcome this challenge, in this work we micro-engineered a LaB6 nanowire-based electron source that emitted a highly collimated electron beam with good lateral and angular alignment. We integrated a passive collimator structure into the support needle tip for the LaB6 nanowire emitter. The collimator formed an axially symmetric electric field around the emission tip of the nanowire. Furthermore, by means of micromanipulation, the support needle tip was bent to align the emitted electron beam with the emitter axis. After installation in an aberration-corrected transmission electron microscope, we characterized the performance of the electron source in a vacuum of 10-8 Pa and achieved atomic resolution in both broad-beam and probe-forming modes at 60 kV beam energy. The natural, unmonochromated 0.20 eV electron energy loss spectroscopy resolution, 20% probe-forming efficiency and 0.4% probe current peak-to-peak noise ratio paired with modest vacuum requirements make the LaB6 nanowire-based electron source an attractive alternative to the standard W-based sources for low-cost electron beam instruments.
    DOI:  https://doi.org/10.1038/s41565-021-00999-w
  9. Sci Rep. 2021 Nov 08. 11(1): 21787
    Room temperature XFEL crystallography reveals asymmetry in the vicinity of the two phylloquinones in photosystem I.
    Stephen M Keable, Adrian Kölsch, Philipp S Simon, Medhanjali Dasgupta, Ruchira Chatterjee, Senthil Kumar Subramanian, Rana Hussein, Mohamed Ibrahim, In-Sik Kim, Isabel Bogacz, Hiroki Makita, Cindy C Pham, Franklin D Fuller, Sheraz Gul, Daniel Paley, Louise Lassalle, Kyle D Sutherlin, Asmit Bhowmick, Nigel W Moriarty, Iris D Young, Johannes P Blaschke, Casper de Lichtenberg, Petko Chernev, Mun Hon Cheah, Sehan Park, Gisu Park, Jangwoo Kim, Sang Jae Lee, Jaehyun Park, Kensuke Tono, Shigeki Owada, Mark S Hunter, Alexander Batyuk, Roland Oggenfuss, Mathias Sander, Serhane Zerdane, Dmitry Ozerov, Karol Nass, Henrik Lemke, Roman Mankowsky, Aaron S Brewster, Johannes Messinger, Nicholas K Sauter, Vittal K Yachandra, Junko Yano, Athina Zouni, Jan Kern.
      Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer.
    DOI:  https://doi.org/10.1038/s41598-021-00236-3
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