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- PDB-8of8: Cryo-EM structure of actomyosin-5a-S1 with the full-length lever ... -

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Basic information

Entry
Database: PDB / ID: 8of8
TitleCryo-EM structure of actomyosin-5a-S1 with the full-length lever (nucleotide free)
Components
  • Actin, alpha skeletal muscle
  • Calmodulin-1
  • Unconventional myosin-Va
KeywordsMOTOR PROTEIN / myosin / cytoskeletal motor / myosin-va / myo5a / myosin-5a / S1 / rigor / nucleotide free / apo / lever / 6IQ / actomyosin / actomyosin-5a / actin / actomyosin-va / actin bound
Function / homology
Function and homology information


actomyosin, myosin complex part / establishment of endoplasmic reticulum localization to postsynapse / regulation of postsynaptic cytosolic calcium ion concentration / axo-dendritic protein transport / positive regulation of cellular response to insulin stimulus / melanosome localization / CaMK IV-mediated phosphorylation of CREB / Cam-PDE 1 activation / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Glycogen breakdown (glycogenolysis) ...actomyosin, myosin complex part / establishment of endoplasmic reticulum localization to postsynapse / regulation of postsynaptic cytosolic calcium ion concentration / axo-dendritic protein transport / positive regulation of cellular response to insulin stimulus / melanosome localization / CaMK IV-mediated phosphorylation of CREB / Cam-PDE 1 activation / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Glycogen breakdown (glycogenolysis) / Activation of RAC1 downstream of NMDARs / Reduction of cytosolic Ca++ levels / Sodium/Calcium exchangers / Activation of Ca-permeable Kainate Receptor / CLEC7A (Dectin-1) induces NFAT activation / endoplasmic reticulum localization / Synthesis of IP3 and IP4 in the cytosol / RHO GTPases activate PAKs / Calmodulin induced events / Inactivation, recovery and regulation of the phototransduction cascade / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Calcineurin activates NFAT / eNOS activation / Ion transport by P-type ATPases / Unblocking of NMDA receptors, glutamate binding and activation / Protein methylation / locomotion involved in locomotory behavior / RAF activation / reactive gliosis / melanin metabolic process / VEGFR2 mediated vascular permeability / RAS processing / developmental pigmentation / Ca2+ pathway / presynaptic endocytosis / unconventional myosin complex / FCERI mediated Ca+2 mobilization / Smooth Muscle Contraction / Extra-nuclear estrogen signaling / insulin-responsive compartment / RHO GTPases activate IQGAPs / melanosome transport / RAF/MAP kinase cascade / negative regulation of dopamine secretion / PKA activation / secretory granule localization / actin filament-based movement / vesicle transport along actin filament / Regulation of actin dynamics for phagocytic cup formation / Regulation of MITF-M-dependent genes involved in pigmentation / Platelet degranulation / melanin biosynthetic process / postsynaptic actin cytoskeleton / hair follicle maturation / regulation of exocytosis / melanocyte differentiation / Stimuli-sensing channels / actomyosin / Ion homeostasis / type 3 metabotropic glutamate receptor binding / negative regulation of synaptic transmission, glutamatergic / ATP-dependent protein binding / positive regulation of vascular associated smooth muscle cell migration / odontogenesis / long-chain fatty acid biosynthetic process / myosin complex / insulin secretion / syntaxin-1 binding / intermediate filament / pigmentation / cytoskeletal motor activator activity / organelle localization by membrane tethering / microfilament motor activity / autophagosome membrane docking / mitochondrion-endoplasmic reticulum membrane tethering / regulation of cardiac muscle cell action potential / tropomyosin binding / myosin heavy chain binding / nitric-oxide synthase binding / mesenchyme migration / troponin I binding / dopamine metabolic process / negative regulation of ryanodine-sensitive calcium-release channel activity / filamentous actin / protein phosphatase activator activity / actin filament bundle / exocytosis / cytoskeletal motor activity / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament / adenylate cyclase binding / calyx of Held / skeletal muscle myofibril / catalytic complex / actin monomer binding / detection of calcium ion / regulation of cardiac muscle contraction / photoreceptor outer segment / regulation of ryanodine-sensitive calcium-release channel activity
Similarity search - Function
Myosin 5a, cargo-binding domain / Class V myosin, motor domain / Dilute domain / DIL domain / Dilute domain profile. / DIL / IQ calmodulin-binding motif / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. ...Myosin 5a, cargo-binding domain / Class V myosin, motor domain / Dilute domain / DIL domain / Dilute domain profile. / DIL / IQ calmodulin-binding motif / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / : / Kinesin motor domain superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / EF-hand domain pair / EF-hand, calcium binding motif / ATPase, nucleotide binding domain / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Calmodulin-1 / Actin, alpha skeletal muscle / Unconventional myosin-Va
Similarity search - Component
Biological speciesMus musculus (house mouse)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsGravett, M.S.C. / Klebl, D.P. / Harlen, O.G. / Read, D.J. / Harris, S.A. / Muench, S.P. / Peckham, M.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Wellcome Trust102174/B/13/Z United Kingdom
Wellcome Trust223125/Z/21/Z United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
Engineering and Physical Sciences Research CouncilEP/T022167/1 United Kingdom
Engineering and Physical Sciences Research CouncilEP/R013012 United Kingdom
CitationJournal: To Be Published
Title: Unveiling the Structural Dynamics of Myosin 5a: Cryo-EM Insights into the Motor Protein's Lever Conformations and Walking Mechanics
Authors: Gravett, M.S.C. / Klebl, D.P. / Harlen, O.G. / Read, D.J. / Harris, S.A. / Muench, S.P. / Peckham, M.
History
DepositionMar 14, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Calmodulin-1
B: Calmodulin-1
C: Calmodulin-1
D: Calmodulin-1
E: Calmodulin-1
F: Calmodulin-1
J: Actin, alpha skeletal muscle
K: Actin, alpha skeletal muscle
L: Actin, alpha skeletal muscle
M: Unconventional myosin-Va


Theoretical massNumber of molelcules
Total (without water)332,55110
Polymers332,55110
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Calmodulin-1


Mass: 16721.350 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Calm1, Calm, Cam, Cam1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0DP26
#2: Protein Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41875.633 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: HIC is tele-methylhistidine / Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle / References: UniProt: P68135
#3: Protein Unconventional myosin-Va / Dilute myosin heavy chain / non-muscle


Mass: 106596.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Myo5a, Dilute / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q99104
Has ligand of interestN

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Myosin-5a bound to F-actinCOMPLEXall0MULTIPLE SOURCES
2Actin, alpha skeletal muscleCOMPLEX#21NATURAL
3Unconventional myosin-Va with calmodulin-1 boundCOMPLEX#31RECOMBINANT
4Calmodulin-1COMPLEX#11RECOMBINANT1 calmodulin bound per IQ domain (6 overall)
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.33 MDaNO
2115 kDa/nmNO
310.11 MDaNO
510.02 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDOrgan
22Oryctolagus cuniculus (rabbit)9986skeletal muscle
33Mus musculus (house mouse)10090
54Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
33Spodoptera frugiperda (fall armyworm)7108
54Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium chlorideKCl1
20.1 mMegtazic acid (EGTA)C14H24N2O101
31 mMmagnesium chlorideMgCl21
410 mM3-morpholinopropane-1-sulfonic acid (MOPS)C7H15NO4S1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 5.26 mg/mL Actin, alpha skeletal muscle 266 mg/mL FlAG-tagged unconventional myosin-Va (residues 1-907) 84.2 mg/mL Calmodulin-1
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 281.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Nominal defocus max: 3600 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 63.13 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4285

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionmanual picking helical start end coordinates
4Gctf1.18CTF correction
7ISOLDEmodel fitting
8Ambermodel fitting
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 838213
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 22337 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeChain-IDDetailsInitial refinement model-IDSource nameType
17PLUDchain D was used as a template to model the motor domain1SwissModelin silico model
2the lever structure with CaM bound was predicted in AlphaFoldAlphaFoldin silico model
37PLU

7plu

7PLUchains J-L were used to model actin3PDBexperimental model

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