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- PDB-6c1h: High-Resolution Cryo-EM Structures of Actin-bound Myosin States R... -

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Entry
Database: PDB / ID: 6c1h
TitleHigh-Resolution Cryo-EM Structures of Actin-bound Myosin States Reveal the Mechanism of Myosin Force Sensing
Components
  • Actin, alpha skeletal muscle
  • Calmodulin
  • Unconventional myosin-Ib
KeywordsSTRUCTURAL PROTEIN / Mechanochemistry / Mechanobiology / Structural Biology / Cytoskeleton / Molecular Motor / Myosin-I
Function / homology
Function and homology information


actin filament-based movement / transferrin transport / vesicle transport along actin filament / CaM pathway / Sodium/Calcium exchangers / Cam-PDE 1 activation / Reduction of cytosolic Ca++ levels / microfilament motor activity / Calmodulin induced events / microfilament motor activity => GO:0000146 ...actin filament-based movement / transferrin transport / vesicle transport along actin filament / CaM pathway / Sodium/Calcium exchangers / Cam-PDE 1 activation / Reduction of cytosolic Ca++ levels / microfilament motor activity / Calmodulin induced events / microfilament motor activity => GO:0000146 / => GO:0120081 / Glycogen breakdown (glycogenolysis) / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / myosin complex / CLEC7A (Dectin-1) induces NFAT activation / Loss of phosphorylation of MECP2 at T308 / Activation of Ca-permeable Kainate Receptor / glycogen catabolic process / PKA activation / CREB1 phosphorylation through the activation of Adenylate Cyclase / mesenchyme migration / establishment of protein localization to mitochondrial membrane / organelle localization by membrane tethering / CaMK IV-mediated phosphorylation of CREB / regulation of cardiac muscle cell action potential / negative regulation of high voltage-gated calcium channel activity / N-terminal myristoylation domain binding / tropomyosin binding / autophagosome membrane docking / mitochondrion-endoplasmic reticulum membrane tethering / myosin heavy chain binding / Phase 0 - rapid depolarisation / Activation of RAC1 downstream of NMDARs / troponin I binding / type 3 metabotropic glutamate receptor binding / Ion transport by P-type ATPases / regulation of synaptic vesicle endocytosis / regulation of cell communication by electrical coupling involved in cardiac conduction / Negative regulation of NMDA receptor-mediated neuronal transmission / negative regulation of peptidyl-threonine phosphorylation / Unblocking of NMDA receptors, glutamate binding and activation / protein phosphatase activator activity / brush border / positive regulation of ryanodine-sensitive calcium-release channel activity / Synthesis of IP3 and IP4 in the cytosol / skeletal muscle thin filament assembly / actin filament bundle / striated muscle thin filament / filamentous actin / regulation of rhodopsin mediated signaling pathway / Smooth Muscle Contraction / response to corticosterone / catalytic complex / RHO GTPases activate PAKs / positive regulation of cyclic-nucleotide phosphodiesterase activity / Activation of AMPK downstream of NMDARs / adenylate cyclase binding / Long-term potentiation / inositol phosphate metabolic process / detection of calcium ion / phosphatidylinositol-3,4,5-trisphosphate binding / actin filament bundle assembly / actin monomer binding / Uptake and function of anthrax toxins / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / cytoskeletal motor activity / Protein methylation / skeletal muscle fiber development / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / DARPP-32 events / skeletal muscle myofibril / adenylate cyclase activator activity / RHO GTPases activate IQGAPs / post-Golgi vesicle-mediated transport / calcium channel inhibitor activity / nitric-oxide synthase binding / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / positive regulation of phosphoprotein phosphatase activity / activation of adenylate cyclase activity / Inactivation, recovery and regulation of the phototransduction cascade / Ion homeostasis / stress fiber / trans-Golgi network membrane / voltage-gated potassium channel complex / regulation of heart rate / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / Wnt signaling pathway, calcium modulating pathway / titin binding / tetrahydrobiopterin metabolic process / substantia nigra development / eNOS activation / sarcomere / actin filament polymerization / positive regulation of protein dephosphorylation / calcium channel complex / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / phosphatidylinositol 3-kinase binding / regulation of ryanodine-sensitive calcium-release channel activity
Similarity search - Function
Class I myosin tail homology domain / Unconventional myosin tail, actin- and lipid-binding / Class I myosin tail homology (TH1) domain profile. / Class I myosin, motor domain / Actin; Chain A, domain 2 / Actin; Chain A, domain 2 / IQ calmodulin-binding motif / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head (motor domain) / Myosin head, motor domain ...Class I myosin tail homology domain / Unconventional myosin tail, actin- and lipid-binding / Class I myosin tail homology (TH1) domain profile. / Class I myosin, motor domain / Actin; Chain A, domain 2 / Actin; Chain A, domain 2 / IQ calmodulin-binding motif / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head (motor domain) / Myosin head, motor domain / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / IQ motif, EF-hand binding site / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / Kinesin motor domain superfamily / ATPase, nucleotide binding domain / EF-hand domain pair / ATPase, nucleotide binding domain / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand calcium-binding domain. / EF-hand domain pair / Nucleotidyltransferase; domain 5 / Roll / Alpha-Beta Complex / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Actin, alpha skeletal muscle / ADENOSINE-5'-DIPHOSPHATE / Calmodulin-1 / Unconventional myosin-Ib
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Rattus norvegicus (Norway rat)
unidentified (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsMentes, A. / Huehn, A. / Liu, X. / Zwolak, A. / Dominguez, R. / Shuman, H. / Ostap, E.M. / Sindelar, C.V.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R37 GM057247 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing.
Authors: Ahmet Mentes / Andrew Huehn / Xueqi Liu / Adam Zwolak / Roberto Dominguez / Henry Shuman / E Michael Ostap / Charles V Sindelar /
Abstract: Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the ...Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.
History
DepositionJan 4, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2018Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 28, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
P: Unconventional myosin-Ib
R: Calmodulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)312,43617
Polymers310,1787
Non-polymers2,25810
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 41862.613 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Protein Unconventional myosin-Ib / Myosin I alpha / MMIa / Myosin heavy chain myr 1


Mass: 84143.930 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat) / References: UniProt: Q05096
#3: Protein Calmodulin /


Mass: 16721.350 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) unidentified (others) / References: UniProt: P0DP23*PLUS
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Complex of actin, myosin-1b, and calmodulin with ADP / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: unidentified (others)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 11 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -167.4 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62000
Details: Resolution estimated by post-processing in RELION using a mask with soft edges that included only the central subunit.
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00522338
ELECTRON MICROSCOPYf_angle_d0.8830248
ELECTRON MICROSCOPYf_dihedral_angle_d8.93513513
ELECTRON MICROSCOPYf_chiral_restr0.0563338
ELECTRON MICROSCOPYf_plane_restr0.0073910

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