bims-strubi Biomed News
on Advances in structural biology
Issue of 2021‒12‒26
fifteen papers selected by
Alessandro Grinzato
European Synchrotron Radiation Facility
  1. Frozen motion: how cryo-EM changes the way we look at ABC transporters.
  2. Machine Learning for Structure Determination in Single-Particle Cryo-Electron Microscopy: A Systematic Review.
  3. Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport.
  4. Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.
  5. High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803.
  6. Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter.
  7. Activation mechanism of PINK1.
  8. A modular platform for automated cryo-FIB workflows.
  9. Caging NLRP3 tames inflammasome activity.
  10. Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus.
  11. Structure of Hsp90-Hsp70-Hop-GR reveals the Hsp90 client-loading mechanism.
  12. Conformational dynamics of the Beta and Kappa SARS-CoV-2 spike proteins and their complexes with ACE2 receptor revealed by cryo-EM.
  13. Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2.
  14. Structure and ion-release mechanism of PIB-4-type ATPases.
  15. Optimization of the In Situ Biasing FIB Sample Preparation for Hafnia-Based Ferroelectric Capacitor.
  1. Trends Biochem Sci. 2021 Dec 17. pii: S0968-0004(21)00247-4. [Epub ahead of print]
    Frozen motion: how cryo-EM changes the way we look at ABC transporters.
    Dmitry Shvarev, Dovile Januliene, Arne Moeller.
      ATP-binding cassette (ABC) transporters are widely present molecular machines that transfer substrates across the cell membrane. ABC transporters are involved in numerous physiological processes and are often clinical targets. Structural biology is fundamental to obtain the molecular details underlying ABC transporter function and suggest approaches to modulate it. Until recently, X-ray crystallography has been the only method capable of providing high-resolution structures of ABC transporters. However, modern cryo-electron microscopy (cryo-EM) opens entirely new ways of studying these dynamic membrane proteins. Cryo-EM enables analyses of targets that resist X-ray crystallography, challenging multicomponent complexes, and the exploration of conformational dynamics. These unique capacities have turned cryo-EM into the dominant technique for structural studies of membrane proteins, including ABC transporters.
    Keywords:  ABC transporters; conformational spectrum; cryo-EM; macromolecular complexes; membrane proteins; structural biology
    DOI:  https://doi.org/10.1016/j.tibs.2021.11.008
  2. IEEE Trans Neural Netw Learn Syst. 2021 Dec 21. PP
    Machine Learning for Structure Determination in Single-Particle Cryo-Electron Microscopy: A Systematic Review.
    Jia-Geng Wu, Yang Yan, Dong-Xu Zhang, Bo-Wen Liu, Qing-Bing Zheng, Xiao-Liang Xie, Shi-Qi Liu, Sheng-Xiang Ge, Zeng-Guang Hou, Ning-Shao Xia.
      Recently, single-particle cryo-electron microscopy (cryo-EM) has become an indispensable method for determining macromolecular structures at high resolution to deeply explore the relevant molecular mechanism. Its recent breakthrough is mainly because of the rapid advances in hardware and image processing algorithms, especially machine learning. As an essential support of single-particle cryo-EM, machine learning has powered many aspects of structure determination and greatly promoted its development. In this article, we provide a systematic review of the applications of machine learning in this field. Our review begins with a brief introduction of single-particle cryo-EM, followed by the specific tasks and challenges of its image processing. Then, focusing on the workflow of structure determination, we describe relevant machine learning algorithms and applications at different steps, including particle picking, 2-D clustering, 3-D reconstruction, and other steps. As different tasks exhibit distinct characteristics, we introduce the evaluation metrics for each task and summarize their dynamics of technology development. Finally, we discuss the open issues and potential trends in this promising field.
    DOI:  https://doi.org/10.1109/TNNLS.2021.3131325
  3. Nat Chem Biol. 2022 Jan;18(1): 101-108
    Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport.
    Deanna L Mendez, Ethan P Lowder, Dustin E Tillman, Molly C Sutherland, Andrea L Collier, Michael J Rau, James A J Fitzpatrick, Robert G Kranz.
      Although the individual structures and respiratory functions of cytochromes are well studied, the structural basis for their assembly, including transport of heme for attachment, are unknown. We describe cryo-electron microscopy (cryo-EM) structures of CcsBA, a bifunctional heme transporter and cytochrome c (cyt c) synthase. Models built from the cryo-EM densities show that CcsBA is trapped with heme in two conformations, herein termed the closed and open states. The closed state has heme located solely at a transmembrane (TM) site, with a large periplasmic domain oriented such that access of heme to the cytochrome acceptor is denied. The open conformation contains two heme moieties, one in the TM-heme site and another in an external site (P-heme site). The presence of heme in the periplasmic site at the base of a chamber induces a large conformational shift that exposes the heme for reaction with apocytochrome c (apocyt c). Consistent with these structures, in vivo and in vitro cyt c synthase studies suggest a mechanism for transfer of the periplasmic heme to cytochrome.
    DOI:  https://doi.org/10.1038/s41589-021-00935-y
  4. Nature. 2021 Dec 22.
    Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.
    Chari M Noddings, Ray Yu-Ruei Wang, Jill L Johnson, David A Agard.
      Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins1-3. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity4-9. GR ligand binding was previously shown to nr inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p2310. However, a molecular understanding of the chaperone-mediated remodelling that occurs between the inactive Hsp70-Hsp90 'client-loading complex' and an activated Hsp90-p23 'client-maturation complex' is lacking for any client, including GR. Here we present a cryo-electron microscopy (cryo-EM) structure of the human GR-maturation complex (GR-Hsp90-p23), revealing that the GR ligand-binding domain is restored to a folded, ligand-bound conformation, while being simultaneously threaded through the Hsp90 lumen. In addition, p23 directly stabilizes native GR using a C-terminal helix, resulting in enhanced ligand binding. This structure of a client bound to Hsp90 in a native conformation contrasts sharply with the unfolded kinase-Hsp90 structure11. Thus, aided by direct co-chaperone-client interactions, Hsp90 can directly dictate client-specific folding outcomes. Together with the GR-loading complex structure12, we present the molecular mechanism of chaperone-mediated GR remodelling, establishing the first, to our knowledge, complete chaperone cycle for any Hsp90 client.
    DOI:  https://doi.org/10.1038/s41586-021-04236-1
  5. Proc Natl Acad Sci U S A. 2022 Jan 04. pii: e2116765118. [Epub ahead of print]119(1):
    High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803.
    Christopher J Gisriel, Jimin Wang, Jinchan Liu, David A Flesher, Krystle M Reiss, Hao-Li Huang, Ke R Yang, William H Armstrong, M R Gunner, Victor S Batista, Richard J Debus, Gary W Brudvig.
      Photosystem II (PSII) enables global-scale, light-driven water oxidation. Genetic manipulation of PSII from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 has provided insights into the mechanism of water oxidation; however, the lack of a high-resolution structure of oxygen-evolving PSII from this organism has limited the interpretation of biophysical data to models based on structures of thermophilic cyanobacterial PSII. Here, we report the cryo-electron microscopy structure of PSII from Synechocystis sp. PCC 6803 at 1.93-Å resolution. A number of differences are observed relative to thermophilic PSII structures, including the following: the extrinsic subunit PsbQ is maintained, the C terminus of the D1 subunit is flexible, some waters near the active site are partially occupied, and differences in the PsbV subunit block the Large (O1) water channel. These features strongly influence the structural picture of PSII, especially as it pertains to the mechanism of water oxidation.
    Keywords:  PsbQ; oxygen-evolving complex; photosynthesis; photosystem II; water oxidation
    DOI:  https://doi.org/10.1073/pnas.2116765118
  6. Nat Chem Biol. 2021 Dec 20.
    Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter.
    Tarjani M Thaker, Smriti Mishra, Wenchang Zhou, Michael Mohan, Qingyu Tang, José D Faraldo-Goméz, Hassane S Mchaourab, Thomas M Tomasiak.
      Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.
    DOI:  https://doi.org/10.1038/s41589-021-00936-x
  7. Nature. 2021 Dec 21.
    Activation mechanism of PINK1.
    Zhong Yan Gan, Sylvie Callegari, Simon A Cobbold, Thomas R Cotton, Michael J Mlodzianoski, Alexander F Schubert, Niall D Geoghegan, Kelly L Rogers, Andrew Leis, Grant Dewson, Alisa Glukhova, David Komander.
      Mutations in the protein kinase PINK1 lead to defects in mitophagy and cause autosomal recessive early onset Parkinson's Disease (EOPD)1,2. PINK1 has many unique features that enable it to phosphorylate ubiquitin and the ubiquitin-like domain of Parkin3-9. Structural analysis of PINK1 from diverse insect species10-12 with and without ubiquitin provided snapshots of distinct structural states yet did not explain how PINK1 is activated. We here elucidate the activation mechanism of PINK1 by crystallography and cryo-EM. A crystal structure of unphosphorylated Pediculus humanus corporis (Ph) PINK1 resolves a previously omitted N-terminal helix revealing how unphosphorylated yet active PINK1 is oriented on mitochondria. We further reveal a 2.35 Å cryo-EM structure of a symmetric PhPINK1 dimer trapped during the process of trans-autophosphorylation, and a 3.1 Å cryo-EM structure of phosphorylated PhPINK1 in the process of undergoing a conformational change to become an active ubiquitin kinase. Structures and phosphorylation studies further identify a role for regulatory PINK1 oxidation. Together, our work delineates the complete activation mechanism of PINK1, illuminates how PINK1 interacts with the mitochondrial outer membrane, and reveals how PINK1 activity may be modulated by mitochondrial reactive oxygen species.
    DOI:  https://doi.org/10.1038/s41586-021-04340-2
  8. Elife. 2021 Dec 24. pii: e70506. [Epub ahead of print]10
    A modular platform for automated cryo-FIB workflows.
    Sven Klumpe, Herman K H Fung, Sara K Goetz, Ievgeniia Zagoriy, Bernhard Hampoelz, Xiaojie Zhang, Philipp S Erdmann, Janina Baumbach, Christoph W Müller, Martin Beck, Jürgen M Plitzko, Julia Mahamid.
      Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.
    Keywords:  D. melanogaster; S. cerevisiae; chlamydomonas reinhardtii; human; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.70506
  9. Cell. 2021 Dec 22. pii: S0092-8674(21)01385-4. [Epub ahead of print]184(26): 6224-6226
    Caging NLRP3 tames inflammasome activity.
    Kate Schroder, Rebecca C Coll.
      How the danger sensor NLRP3 is activated is intensively debated. Using cryo-electron microscopy (EM) approaches, Andreeva and colleagues made the remarkable discovery that inactive NLRP3 forms a double ring of 12-16 monomers that shield its pyrin domains from the cytosol. We discuss this surprising new mechanism of inflammasome regulation.
    DOI:  https://doi.org/10.1016/j.cell.2021.11.035
  10. EMBO J. 2021 Dec 22. e109728
    Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus.
    Michaela J Conley, Judith M Short, Andrew M Burns, James Streetley, Joshua Hutchings, Saskia E Bakker, B Joanne Power, Hussain Jaffery, Joanne Haney, Giulia Zanetti, Pablo R Murcia, Murray Stewart, Rachel Fearns, Swetha Vijayakrishnan, David Bhella.
      Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.
    Keywords:  cryo-EM; cryo-ET; glycoprotein; matrix protein; virus structure
    DOI:  https://doi.org/10.15252/embj.2021109728
  11. Nature. 2021 Dec 22.
    Structure of Hsp90-Hsp70-Hop-GR reveals the Hsp90 client-loading mechanism.
    Ray Yu-Ruei Wang, Chari M Noddings, Elaine Kirschke, Alexander G Myasnikov, Jill L Johnson, David A Agard.
      Maintaining a healthy proteome is fundamental for the survival of all organisms1. Integral to this are Hsp90 and Hsp70, molecular chaperones that together facilitate the folding, remodelling and maturation of the many 'client proteins' of Hsp902. The glucocorticoid receptor (GR) is a model client protein that is strictly dependent on Hsp90 and Hsp70 for activity3-7. Chaperoning GR involves a cycle of inactivation by Hsp70; formation of an inactive GR-Hsp90-Hsp70-Hop 'loading' complex; conversion to an active GR-Hsp90-p23 'maturation' complex; and subsequent GR release8. However, to our knowledge, a molecular understanding of this intricate chaperone cycle is lacking for any client protein. Here we report the cryo-electron microscopy structure of the GR-loading complex, in which Hsp70 loads GR onto Hsp90, uncovering the molecular basis of direct coordination by Hsp90 and Hsp70. The structure reveals two Hsp70 proteins, one of which delivers GR and the other scaffolds the Hop cochaperone. Hop interacts with all components of the complex, including GR, and poises Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized through an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR-maturation complex structure9, we present a complete molecular mechanism of chaperone-dependent client remodelling, and establish general principles of client recognition, inhibition, transfer and activation.
    DOI:  https://doi.org/10.1038/s41586-021-04252-1
  12. Nat Commun. 2021 12 20. 12(1): 7345
    Conformational dynamics of the Beta and Kappa SARS-CoV-2 spike proteins and their complexes with ACE2 receptor revealed by cryo-EM.
    Yifan Wang, Cong Xu, Yanxing Wang, Qin Hong, Chao Zhang, Zuyang Li, Shiqi Xu, Qinyu Zuo, Caixuan Liu, Zhong Huang, Yao Cong.
      The emergence of SARS-CoV-2 Kappa and Beta variants with enhanced transmissibility and resistance to neutralizing antibodies has created new challenges for the control of the ongoing COVID-19 pandemic. Understanding the structural nature of Kappa and Beta spike (S) proteins and their association with ACE2 is of significant importance. Here we present two cryo-EM structures for each of the Kappa and Beta spikes in the open and open-prone transition states. Compared with wild-type (WT) or G614 spikes, the two variant spikes appear more untwisted/open especially for Beta, and display a considerable population shift towards the open state as well as more pronounced conformational dynamics. Moreover, we capture four conformational states of the S-trimer/ACE2 complex for each of the two variants, revealing an enlarged conformational landscape for the Kappa and Beta S-ACE2 complexes and pronounced population shift towards the three RBDs up conformation. These results implicate that the mutations in Kappa and Beta may modify the kinetics of receptor binding and viral fusion to improve virus fitness. Combined with biochemical analysis, our structural study shows that the two variants are enabled to efficiently interact with ACE2 receptor despite their sensitive ACE2 binding surface is modified to escape recognition by some potent neutralizing MAbs. Our findings shed new light on the pathogenicity and immune evasion mechanism of the Beta and Kappa variants.
    DOI:  https://doi.org/10.1038/s41467-021-27350-0
  13. Elife. 2021 Dec 23. pii: e74707. [Epub ahead of print]10
    Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2.
    Juan Feng, Xianchi Dong, Adam DeCosta, Yang Su, Fiona Angrisano, Katarzyna A Sala, Andrew M Blagborough, Chafen Lu, Timothy A Springer.
      HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.
    Keywords:  infectious disease; microbiology; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.74707
  14. Elife. 2021 Dec 24. pii: e73124. [Epub ahead of print]10
    Structure and ion-release mechanism of PIB-4-type ATPases.
    Christina Grønberg, Qiaoxia Hu, Dhani Ram Mahato, Elena Longhin, Nina Salustros, Annette Duelli, Pin Lyu, Viktoria Bågenholm, Jonas Eriksson, Komal Umashankar Rao, Domhnall Iain Henderson, Gabriele Meloni, Magnus Andersson, Tristan Croll, Gabriela Godaly, Kaituo Wang, Pontus Gourdon.
      Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here we present structures and complementary functional analyses of an archetypal PIB‑4‑ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy metal binding domains, and provides fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turn-over of PIB‑ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in e.g. drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.
    Keywords:  biochemistry; chemical biology; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.73124
  15. Micromachines (Basel). 2021 Nov 24. pii: 1436. [Epub ahead of print]12(12):
    Optimization of the In Situ Biasing FIB Sample Preparation for Hafnia-Based Ferroelectric Capacitor.
    Qilan Zhong, Yiwei Wang, Yan Cheng, Zhaomeng Gao, Yunzhe Zheng, Tianjiao Xin, Yonghui Zheng, Rong Huang, Hangbing Lyu.
      Hafnia-based ferroelectric (FE) thin films have received extensive attention in both academia and industry, benefitting from their outstanding scalability and excellent CMOS compatibility. Hafnia-based FE capacitors in particular have the potential to be used in dynamic random-access memory (DRAM) applications. Obtaining fine structure characterization at ultra-high spatial resolution is helpful for device performance optimization. Hence, sample preparation by the focused ion beam (FIB) system is an essential step, especially for in situ biasing experiments in a transmission electron microscope (TEM). In this work, we put forward three tips to improve the success rate of in situ biasing experiments: depositing a carbon protective layer to position the interface, welding the sample on the top of the Cu column of the TEM grid, and cutting the sample into a comb-like shape. By these means, in situ biasing of the FE capacitor was realized in TEM, and electric-field-induced tetragonal (t-) to monoclinic (m-) structure transitions in Hf0.5Zr0.5O2 FE film were observed. The improvement of FIB sample preparation technology can greatly enhance the quality of in situ biasing TEM samples, improve the success rate, and extend from capacitor sample preparation to other types.
    Keywords:  FIB sample preparation technology; hafnia-based ferroelectric; in situ biasing
    DOI:  https://doi.org/10.3390/mi12121436
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