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 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.

scholarly journals Elasticity, friction, and pathway of γ-subunit rotation in FoF1-ATP synthase

2015 ◽  
Vol 112 (34) ◽  
pp. 10720-10725 ◽  
Author(s):  
Kei-ichi Okazaki ◽  
Gerhard Hummer
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Viscoelastic Model ◽  
Thermodynamic Efficiency ◽  
Γ Subunit ◽  
Frictional Dissipation ◽  
Rotary Motors ◽  
Subunit Rotation ◽  
Dynamics Simulations ◽  
Rotary Motor

We combine molecular simulations and mechanical modeling to explore the mechanism of energy conversion in the coupled rotary motors of FoF1-ATP synthase. A torsional viscoelastic model with frictional dissipation quantitatively reproduces the dynamics and energetics seen in atomistic molecular dynamics simulations of torque-driven γ-subunit rotation in the F1-ATPase rotary motor. The torsional elastic coefficients determined from the simulations agree with results from independent single-molecule experiments probing different segments of the γ-subunit, which resolves a long-lasting controversy. At steady rotational speeds of ∼1 kHz corresponding to experimental turnover, the calculated frictional dissipation of less than kBT per rotation is consistent with the high thermodynamic efficiency of the fully reversible motor. Without load, the maximum rotational speed during transitions between dwells is reached at ∼1 MHz. Energetic constraints dictate a unique pathway for the coupled rotations of the Fo and F1 rotary motors in ATP synthase, and explain the need for the finer stepping of the F1 motor in the mammalian system, as seen in recent experiments. Compensating for incommensurate eightfold and threefold rotational symmetries in Fo and F1, respectively, a significant fraction of the external mechanical work is transiently stored as elastic energy in the γ-subunit. The general framework developed here should be applicable to other molecular machines.

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.

Cryo-EM structure of the mammalian ATP synthase tetramer bound with inhibitory protein IF1

Science ◽  
2019 ◽  
Vol 364 (6445) ◽  
pp. 1068-1075 ◽  
Author(s):  
Jinke Gu ◽  
Laixing Zhang ◽  
Shuai Zong ◽  
Runyu Guo ◽  
Tianya Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atp Synthase ◽  
Mammalian Cells ◽  
Low Ph ◽  
Inhibitory Protein ◽  
Cryo Electron Microscopy ◽  
The Matrix ◽  
Microscopy Method ◽  
Electron Microscopy Method

The mitochondrial adenosine triphosphate (ATP) synthase produces most of the ATP required by mammalian cells. We isolated porcine tetrameric ATP synthase and solved its structure at 6.2-angstrom resolution using a single-particle cryo–electron microscopy method. Two classical V-shaped ATP synthase dimers lie antiparallel to each other to form an H-shaped ATP synthase tetramer, as viewed from the matrix. ATP synthase inhibitory factor subunit 1 (IF1) is a well-known in vivo inhibitor of mammalian ATP synthase at low pH. Two IF1 dimers link two ATP synthase dimers, which is consistent with the ATP synthase tetramer adopting an inhibited state. Within the tetramer, we refined structures of intact ATP synthase in two different rotational conformations at 3.34- and 3.45-Å resolution.

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.

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

2021 ◽  
Author(s):  
Brandon Malone ◽  
James Chen ◽  
Qi Wang ◽  
Eliza Llewellyn ◽  
Young Joo Choi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Rna Polymerase ◽  
Critical Role ◽  
Structural Basis ◽  
Rna Dependent Rna Polymerase ◽  
Present Evidence ◽  
Cryo Electron Microscopy ◽  
Dynamics Simulations ◽  
Transcription And Replication

AbstractBacktracking, 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 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 crosslinking, 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 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.Significance StatementThe COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backwards motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals.

scholarly journals Mechanisms of Strain Diversity of Disease-Associated in-Register Parallel β-Sheet Amyloids and Implications About Prion Strains

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 110 ◽  
Author(s):  
Yuzuru Taguchi ◽  
Hiroki Otaki ◽  
Noriyuki Nishida
Keyword(s):  
Electron Microscopy ◽  
Resonance Spectroscopy ◽  
Post Translational Modifications ◽  
Prion Strain ◽  
Local Structures ◽  
Strain Diversity ◽  
Cryo Electron Microscopy ◽  
Prion Strains ◽  
Dynamics Simulations ◽  
Β Sheet

The mechanism of prion strain diversity remains unsolved. Investigation of inheritance and diversification of protein-based pathogenic information demands the identification of the detailed structures of abnormal isoforms of the prion protein (PrPSc); however, achieving purification is difficult without affecting infectivity. Similar prion-like properties are recognized also in other disease-associated in-register parallel β-sheet amyloids including Tau and α-synuclein (αSyn) amyloids. Investigations into structures of those amyloids via solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances due to their relatively small sizes and lack of post-translational modifications. Herein, we review advances regarding pathogenic amyloids, particularly Tau and αSyn, and discuss implications about strain diversity mechanisms of prion/PrPSc from the perspective that PrPSc is an in-register parallel β-sheet amyloid. Additionally, we present our recent data of molecular dynamics simulations of αSyn amyloid, which suggest significance of compatibility between β-sheet propensities of the substrate and local structures of the template for stability of amyloid structures. Detailed structures of αSyn and Tau amyloids are excellent models of pathogenic amyloids, including PrPSc, to elucidate strain diversity and pathogenic mechanisms.

scholarly journals Ensemble cryo-electron microscopy reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex

2021 ◽  
Author(s):  
James Chen ◽  
Qi Wang ◽  
Brandon Malone ◽  
Eliza Llewellyn ◽  
Yakov Pechersky ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Active Site ◽  
Rna Synthesis ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Rna Dependent Rna Polymerase ◽  
Conformational States ◽  
Cryo Electron Microscopy ◽  
Allosteric Mechanism ◽  
Dynamics Simulations

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex, or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backwards on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here, we use cryo-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results suggest a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

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