scholarly journals The mechanical inhibition of the isolated Vo from V-ATPase for proton conductance

2020 ◽  
Author(s):  
Jun-ichi Kishikawa ◽  
Atsuko Nakanishi ◽  
Aya Furuta ◽  
Takayuki Kato ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Atomic Resolution ◽  
Converting Enzyme ◽  
Proton Conductance ◽  
Membrane Domain ◽  
Enzyme Coupling ◽  
Cryo Electron Microscopy ◽  
Flow Through ◽  
Energy Converting

AbstractV-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 moiety, with proton flow through the Vo membrane moiety, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo of eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and the isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo adopts the auto-inhibited form and how the holo complex prevents the formation of this inhibited Vo form.One Sentence SummaryCryo-EM structures of rotary V-ATPase reveal the ON-OFF switching mechanism of H+ translocation in the Vo membrane domain.

scholarly journals Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jun-ichi Kishikawa ◽  
Atsuko Nakanishi ◽  
Aya Furuta ◽  
Takayuki Kato ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Atomic Resolution ◽  
Converting Enzyme ◽  
Proton Conductance ◽  
Membrane Domain ◽  
Enzyme Coupling ◽  
Cryo Electron Microscopy ◽  
Flow Through ◽  
Energy Converting

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 domain, with proton flow through the Vo membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo domain adopts the auto-inhibited form and how the holo complex prevents formation of the inhibited Vo form.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

scholarly journals A resolution record for cryoEM

2021 ◽  
Vol 10 ◽  
Author(s):  
Jonathan Ashmore ◽  
Bridget Carragher ◽  
Peter B Rosenthal ◽  
William Weis
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Structure Determination ◽  
Test Specimen ◽  
Structural Information ◽  
Atomic Resolution ◽  
Fast Growing ◽  
Cryo Electron Microscopy ◽  
New Instrument ◽  
Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

Forge Wednesday Roundup - June 10, 2020

2021 ◽  
Author(s):  
H.L. Cu Si
Keyword(s):  
Electron Microscopy ◽  
Facial Recognition ◽  
Academic Research ◽  
Atomic Resolution ◽  
Rapa Nui ◽  
Social Distancing ◽  
Cryo Electron Microscopy

In today’s Roundup: COVID is still with us; pair of papers quantifies effects of social distancing; IBM bails on facial recognition, warns of harms; questioning “collapse” of Rapa Nui; attention turns to antibodies for COVID-19 therapies; WHO walks back confusing statement on asymptomatic COVID transmission; cryo-electron microscopy reveals proteins at atomic resolution; unstoppable newsbot runs amok, propagates bias; exploring the lure of misinformation; academic research goes on strike against racism; much more...

scholarly journals Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display

2019 ◽  
Vol 5 (9) ◽  
pp. eaaw2853 ◽  
Author(s):  
Charles Vragniau ◽  
Joshua C. Bufton ◽  
Frédéric Garzoni ◽  
Emilie Stermann ◽  
Fruzsina Rabi ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Electron Microscopy ◽  
High Performance ◽  
Vaccine Candidate ◽  
Cost Effective ◽  
Atomic Resolution ◽  
Self Assembling ◽  
Cryo Electron Microscopy ◽  
Architectural Features ◽  
Multimeric Protein

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo–electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.

scholarly journals Atomic Resolution Structures of Human Bufaviruses Determined by Cryo-Electron Microscopy

Viruses ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 22 ◽  
Author(s):  
Maria Ilyas ◽  
Mario Mietzsch ◽  
Shweta Kailasan ◽  
Elina Väisänen ◽  
Mengxiao Luo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Resolution ◽  
Cryo Electron Microscopy

scholarly journals Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

2015 ◽  
Vol 112 (43) ◽  
pp. 13237-13242 ◽  
Author(s):  
Lorenzo Sborgi ◽  
Francesco Ravotti ◽  
Venkata P. Dandey ◽  
Mathias S. Dick ◽  
Adam Mazur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Specific Binding ◽  
3D Structure ◽  
High Mobility ◽  
Atomic Resolution ◽  
Receptor Protein ◽  
Filament Formation ◽  
Cryo Electron Microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

scholarly journals Cryo-EM Reveals the Structural Basis of Microtubule Depolymerization by Kinesin-13s

2017 ◽  
Author(s):  
Matthieu P. M. H. Benoit ◽  
Ana B. Asenjo ◽  
Hernando Sosa
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Distinct Group ◽  
Atomic Resolution ◽  
Structural Basis ◽  
Structural Elements ◽  
Microtubule Depolymerization ◽  
Cryo Electron Microscopy ◽  
Kinesin Superfamily ◽  
Motile Activity

SummaryKinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promotes microtubule depolymerization and lacks motile activity. The molecular mechanism by which the kinesins depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue we obtained near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A constructs bound to curved or straight tubulin in different nucleotide states. The structures show how nucleotide induced conformational changes near the catalytic site are coupled with kinesin-13-specific structural elements to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.

scholarly journals Cryo-electron microscopy structure of a nucleosome-bound SWI/SNF chromatin remodeling complex

2019 ◽  
Author(s):  
Yan Han ◽  
Alexis A Reyes ◽  
Sara Malik ◽  
Yuan He
Keyword(s):  
Electron Microscopy ◽  
Chromatin Remodeling ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Gene Activation ◽  
The Body ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Chromatin Remodeling Complex ◽  
High Resolution Structure

AbstractThe multi-subunit chromatin remodeling complex SWI/SNF1–3 is highly conserved from yeast to humans and plays critical roles in various cellular processes including transcription and DNA damage repair4, 5. It uses the energy from ATP hydrolysis to remodel chromatin structure by sliding and evicting the histone octamer6–10, creating DNA regions that become accessible to other essential protein complexes. However, our mechanistic understanding of the chromatin remodeling activity is largely hindered by the lack of a high-resolution structure of any complex from this family. Here we report the first structure of SWI/SNF from the yeast S. cerevisiae bound to a nucleosome at near atomic resolution determined by cryo-electron microscopy (cryo-EM). In the structure, the Arp module is sandwiched between the ATPase and the Body module of the complex, with the Snf2 HSA domain connecting all modules. The HSA domain also extends into the Body and anchors at the opposite side of the complex. The Body contains an assembly scaffold composed of conserved subunits Snf12 (SMARCD/BAF60), Snf5 (SMARCB1/BAF47/ INI1) and an asymmetric dimer of Swi3 (SMARCC/BAF155/170). Another conserved subunit Swi1 (ARID1/BAF250) folds into an Armadillo (ARM) repeat domain that resides in the core of the SWI/SNF Body, acting as a molecular hub. In addition to the interaction between Snf2 and the nucleosome, we also observed interactions between the conserved Snf5 subunit and the histones at the acidic patch, which could serve as an anchor point during active DNA translocation. Our structure allows us to map and rationalize a subset of cancer-related mutations in the human SWI/SNF complex and propose a model of how SWI/SNF recognizes and remodels the +1 nucleosome to generate nucleosome-depleted regions during gene activation11–13.

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