scholarly journals Remodeling and activation mechanisms of outer arm dynein revealed by cryo-EM

2020 ◽  
Author(s):  
Shintaroh Kubo ◽  
Shun Kai Yang ◽  
Corbin Black ◽  
Daniel Dai ◽  
Melissa Valente ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Frequency ◽  
Complex Structure ◽  
Eukaryotic Cells ◽  
Native State ◽  
Ciliary Beating ◽  
Cryo Electron Microscopy ◽  
Activation Mechanisms ◽  
Dynein Arms ◽  
Dynamics Simulations

Cilia are thin microtubule-based protrusions of eukaryotic cells, beating at high frequency to propel the cell in sperms or clear mucus in the respiratory tract. The ciliary beating is driven by the outer arm dynein arms (ODAs) which anchor on the doublet microtubules. Here, we report the ODA complex structure from the native doublet microtubules by cryo-electron microscopy. Our structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with molecular dynamics simulations, we present a model of how the attachment of the ODA complex to the doublet microtubule induces rearrangements and activation mechanisms within the ODA complex.

scholarly journals Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

2016 ◽  
Vol 22 (6) ◽  
pp. 1316-1328 ◽  
Author(s):  
Michael Marko ◽  
Chyongere Hsieh ◽  
Eric Leith ◽  
David Mastronarde ◽  
Sohei Motoki
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Practical Experience ◽  
Phase Plate ◽  
Native State ◽  
Automated Data Collection ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Phase Plates ◽  
Set Up

AbstractPhase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

scholarly journals F0-F1 coupling and symmetry mismatch in ATP synthase resolved in every F0 rotation step

2021 ◽  
Author(s):  
Shintaroh Kubo ◽  
Toru Niina ◽  
Shoji Takada
Keyword(s):  
Electron Microscopy ◽  
Atp Synthase ◽  
Energy Production ◽  
Comprehensive Understanding ◽  
Cellular Energy ◽  
Cryo Electron Microscopy ◽  
Rotary Motors ◽  
Elastic Distortion ◽  
Dynamics Simulations ◽  
Symmetric Structures

The F0F1 ATP synthase, essential for cellular energy production, is composed of the F0 and F1 rotary motors. While both F0 and F1 have pseudo-symmetric structures, their symmetries do not match. How the symmetry mismatch is solved remains elusive due to missing intermediate structures of rotational steps. Here, for ATP synthases with 3- and 10-fold symmetries in F1 and F0, respectively, we uncovered the mechanical couplings between F0 and F1 at every 36° rotation step via molecular dynamics simulations and comparison of cryo-electron microscopy structures from three species. We found that the frustration is shared by several elements. The F1 stator partially rotates relative to the F0 stator via elastic distortion of the b-subunits. The rotor can be distorted. The c-ring rotary angles can be deviated from symmetric ones. Additionally, the F1 motor may take non-canonical structures relieving stronger frustration. Together, we provide comprehensive understanding to solve the symmetry mismatch.

scholarly journals Structural basis for backtracking by the SARS-CoV-2 replication–transcription complex

2021 ◽  
Vol 118 (19) ◽  
pp. e2102516118
Author(s):  
Brandon Malone ◽  
James Chen ◽  
Qi Wang ◽  
Eliza Llewellyn ◽  
Young Joo Choi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Structural Basis ◽  
Nucleoside Triphosphate ◽  
Cross Linking ◽  
Rna Dependent Rna Polymerase ◽  
Present Evidence ◽  
Cryo Electron Microscopy ◽  
Dynamics Simulations ◽  
Transcription And Replication

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA–protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3′ segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3′ end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

scholarly journals Shulin packages axonemal outer dynein arms for ciliary targeting

Science ◽  
2021 ◽  
Vol 371 (6532) ◽  
pp. 910-916
Author(s):  
Girish R. Mali ◽  
Ferdos Abid Ali ◽  
Clinton K. Lau ◽  
Farida Begum ◽  
Jérôme Boulanger ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Motor Activity ◽  
Compact Form ◽  
Unknown Mechanism ◽  
Closed Conformation ◽  
Cryo Electron Microscopy ◽  
Two Factors ◽  
Dynein Arms ◽  
Cilia And Flagella

The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During ciliogenesis, these ~1.8-megadalton complexes are assembled in the cytoplasm and targeted to cilia by an unknown mechanism. Here, we used the ciliateTetrahymenato identify two factors (Q22YU3 and Q22MS1) that bind ODAs in the cytoplasm and are required for ODA delivery to cilia. Q22YU3, which we named Shulin, locked the ODA motor domains into a closed conformation and inhibited motor activity. Cryo–electron microscopy revealed how Shulin stabilized this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

scholarly journals The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ahmad Abdelzaher Zaki Khalifa ◽  
Muneyoshi Ichikawa ◽  
Daniel Dai ◽  
Shintaroh Kubo ◽  
Corbin Steven Black ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Tetrahymena Thermophila ◽  
Building Blocks ◽  
Post Translational Modifications ◽  
Ciliary Beating ◽  
Cryo Electron Microscopy ◽  
The Stability ◽  
And Function ◽  
Β Tubulin ◽  
Junction Proteins

Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.

scholarly journals Atomic model for the dimeric FO region of mitochondrial ATP synthase

Science ◽  
2017 ◽  
Vol 358 (6365) ◽  
pp. 936-940 ◽  
Author(s):  
Hui Guo ◽  
Stephanie A. Bueler ◽  
John L. Rubinstein
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Crystal Structures ◽  
Atp Synthase ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Atomic Model ◽  
Eukaryotic Cells ◽  
Cryo Electron Microscopy ◽  
Mitochondrial Atp Synthase

Mitochondrial adenosine triphosphate (ATP) synthase produces the majority of ATP in eukaryotic cells, and its dimerization is necessary to create the inner membrane folds, or cristae, characteristic of mitochondria. Proton translocation through the membrane-embedded FO region turns the rotor that drives ATP synthesis in the soluble F1 region. Although crystal structures of the F1 region have illustrated how this rotation leads to ATP synthesis, understanding how proton translocation produces the rotation has been impeded by the lack of an experimental atomic model for the FO region. Using cryo–electron microscopy, we determined the structure of the dimeric FO complex from Saccharomyces cerevisiae at a resolution of 3.6 angstroms. The structure clarifies how the protons travel through the complex, how the complex dimerizes, and how the dimers bend the membrane to produce cristae.

Prediction of Protein Complex Structure Using Surface-Induced Dissociation and Cryo-Electron Microscopy

2021 ◽  
Author(s):  
Justin T. Seffernick ◽  
Shane M. Canfield ◽  
Sophie R. Harvey ◽  
Vicki H. Wysocki ◽  
Steffen Lindert
Keyword(s):  
Electron Microscopy ◽  
Protein Complex ◽  
Complex Structure ◽  
Surface Induced Dissociation ◽  
Cryo Electron Microscopy

scholarly journals Cryo-Electron Microscopy Structure of the αIIbβ3-Abciximab Complex

2020 ◽  
Vol 40 (3) ◽  
pp. 624-637 ◽  
Author(s):  
Dragana Nešić ◽  
Yixiao Zhang ◽  
Aleksandar Spasic ◽  
Jihong Li ◽  
Davide Provasi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Binding Pocket ◽  
Antiplatelet Drug ◽  
Potential Contribution ◽  
Variable Regions ◽  
Heavy Chains ◽  
Cryo Electron Microscopy ◽  
Fibrinogen Binding ◽  
Complementarity Determining Regions ◽  
Dynamics Simulations

Objective: The αIIbβ3 antagonist antiplatelet drug abciximab is the chimeric antigen-binding fragment comprising the variable regions of murine monoclonal antibody 7E3 and the constant domains of human IgG1 and light chain κ. Previous mutagenesis studies suggested that abciximab binds to the β3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent β1-α1 helix. These studies could not, however, assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of either the αIIbβ3-binding pocket for fibrinogen or the β3 SDL (which is not part of the binding pocket but affects fibrinogen binding), or some combination of these effects. To address this gap, we used cryo-electron microscopy to determine the structure of the αIIbβ3-abciximab complex at 2.8 Å resolution. Approach and Results: The interacting surface of abciximab is comprised of residues from all 3 complementarity-determining regions of both the light and heavy chains, with high representation of aromatic residues. Binding is primarily to the β3 SDL and neighboring residues, the β1-α1 helix, and β3 residues Ser211, Val212 and Met335. Unexpectedly, the structure also indicated several interactions with αIIb. As judged by the cryo-electron microscopy model, molecular-dynamics simulations, and mutagenesis, the binding of abciximab does not appear to rely on the interaction with the αIIb residues and does not result in disruption of the fibrinogen-binding pocket; it does, however, compress and reduce the flexibility of the SDL. Conclusions: We deduce that abciximab prevents ligand binding by steric interference, with a potential contribution via displacement of the SDL and limitation of the flexibility of the SDL residues.

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