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- EMDB-16850: Cryo-EM structure of actomyosin-5a-S1 with the full-length lever ... -

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Entry
Database: EMDB / ID: EMD-16850
TitleCryo-EM structure of actomyosin-5a-S1 with the full-length lever (nucleotide free, class D)
Map dataDeepEMhancer post-processed map
Sample
  • Complex: Myosin-5a bound to F-actin
    • Complex: Actin, alpha skeletal muscle
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: Unconventional myosin-Va with calmodulin-1 bound
      • Protein or peptide: Unconventional myosin-Va
    • Complex: Calmodulin-1
      • Protein or peptide: Calmodulin-1
Keywordsmyosin / cytoskeletal motor / myosin-va / myo5a / myosin-5a / S1 / rigor / nucleotide free / apo / lever / 6IQ / actomyosin / actomyosin-5a / actin / actomyosin-va / actin bound / MOTOR PROTEIN
Function / homology
Function and homology information


establishment of endoplasmic reticulum localization to postsynapse / regulation of postsynaptic cytosolic calcium ion concentration / 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 / melanosome localization / endoplasmic reticulum localization / Reduction of cytosolic Ca++ levels ...establishment of endoplasmic reticulum localization to postsynapse / regulation of postsynaptic cytosolic calcium ion concentration / 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 / melanosome localization / endoplasmic reticulum localization / Reduction of cytosolic Ca++ levels / Sodium/Calcium exchangers / Activation of Ca-permeable Kainate Receptor / Synthesis of IP3 and IP4 in the cytosol / CLEC7A (Dectin-1) induces NFAT activation / RHO GTPases activate PAKs / Calmodulin induced events / Inactivation, recovery and regulation of the phototransduction cascade / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / eNOS activation / Ion transport by P-type ATPases / Calcineurin activates NFAT / Unblocking of NMDA receptors, glutamate binding and activation / locomotion involved in locomotory behavior / Protein methylation / melanin metabolic process / RAF activation / VEGFR2 mediated vascular permeability / vesicle transport along actin filament / RAS processing / unconventional myosin complex / insulin-responsive compartment / FCERI mediated Ca+2 mobilization / Ca2+ pathway / RHO GTPases activate IQGAPs / Extra-nuclear estrogen signaling / RAF/MAP kinase cascade / PKA activation / developmental pigmentation / melanosome transport / secretory granule localization / Smooth Muscle Contraction / Regulation of MITF-M-dependent genes involved in pigmentation / Regulation of actin dynamics for phagocytic cup formation / actin filament-based movement / melanin biosynthetic process / hair follicle maturation / Platelet degranulation / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / melanocyte differentiation / actomyosin / Stimuli-sensing channels / Ion homeostasis / long-chain fatty acid biosynthetic process / myosin complex / insulin secretion / odontogenesis / intermediate filament / cytoskeletal motor activator activity / organelle localization by membrane tethering / pigmentation / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / microfilament motor activity / myosin heavy chain binding / presynaptic endocytosis / regulation of cardiac muscle cell action potential / tropomyosin binding / negative regulation of ryanodine-sensitive calcium-release channel activity / troponin I binding / filamentous actin / exocytosis / calcineurin-mediated signaling / mesenchyme migration / actin filament bundle / cytoskeletal motor activity / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / protein phosphatase activator activity / adenylate cyclase binding / regulation of ryanodine-sensitive calcium-release channel activity / skeletal muscle thin filament assembly / actin monomer binding / catalytic complex / photoreceptor outer segment / detection of calcium ion / regulation of cardiac muscle contraction / smooth endoplasmic reticulum / calcium channel inhibitor activity / cellular response to interferon-beta / stress fiber / skeletal muscle fiber development / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / vesicle-mediated transport / titin binding / voltage-gated potassium channel complex / actin filament polymerization / sperm midpiece / myelination / calcium channel complex
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 speciesOryctolagus cuniculus (rabbit) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsGravett MSC / Klebl DP / Harlen OG / Read DJ / Harris SA / Muench SP / Peckham M
Funding support United Kingdom, 5 items
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: Structure / Year: 2024
Title: Exploiting cryo-EM structures of actomyosin-5a to reveal the physical properties of its lever.
Authors: Molly S C Gravett / David P Klebl / Oliver G Harlen / Daniel J Read / Stephen P Muench / Sarah A Harris / Michelle Peckham /
Abstract: Myosin 5a (Myo5a) is a dimeric processive motor protein that transports cellular cargos along filamentous actin (F-actin). Its long lever is responsible for its large power-stroke, step size, and ...Myosin 5a (Myo5a) is a dimeric processive motor protein that transports cellular cargos along filamentous actin (F-actin). Its long lever is responsible for its large power-stroke, step size, and load-bearing ability. Little is known about the levers' structure and physical properties, and how they contribute to walking mechanics. Using cryoelectron microscopy (cryo-EM) and molecular dynamics (MD) simulations, we resolved the structure of monomeric Myo5a, comprising the motor domain and full-length lever, bound to F-actin. The range of its lever conformations revealed its physical properties, how stiffness varies along its length and predicts a large, 35 nm, working stroke. Thus, the newly released trail head in a dimeric Myo5a would only need to perform a small diffusive search for its new binding site on F-actin, and stress would only be generated across the dimer once phosphate is released from the lead head, revealing new insight into the walking behavior of Myo5a.
History
DepositionMar 14, 2023-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateApr 9, 2025-
Current statusApr 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16850.png

Downloads & links

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Map

FileDownload / File: emd_16850.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer post-processed map
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
2.13 Å/pix.
x 250 pix.
= 532.5 Å		2.13 Å/pix.
x 250 pix.
= 532.5 Å		2.13 Å/pix.
x 250 pix.
= 532.5 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.13 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.0008946664 - 1.4243865
Average (Standard dev.)0.0014060596 (±0.025409117)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 532.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16850_msk_1.map
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Additional map: Gaussian filtered (SD 5) map

Fileemd_16850_additional_1.map
AnnotationGaussian filtered (SD 5) map
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Additional map: Masked post-processed map

Fileemd_16850_additional_2.map
AnnotationMasked post-processed map
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Additional map: Raw map

Fileemd_16850_additional_3.map
AnnotationRaw map
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Half map: Half map 1

Fileemd_16850_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_16850_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : Myosin-5a bound to F-actin

EntireName: Myosin-5a bound to F-actin
Components
  • Complex: Myosin-5a bound to F-actin
    • Complex: Actin, alpha skeletal muscle
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: Unconventional myosin-Va with calmodulin-1 bound
      • Protein or peptide: Unconventional myosin-Va
    • Complex: Calmodulin-1
      • Protein or peptide: Calmodulin-1

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Supramolecule #1: Myosin-5a bound to F-actin

SupramoleculeName: Myosin-5a bound to F-actin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 20 KDa

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Supramolecule #2: Actin, alpha skeletal muscle

SupramoleculeName: Actin, alpha skeletal muscle / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Organ: skeletal muscle

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Supramolecule #3: Unconventional myosin-Va with calmodulin-1 bound

SupramoleculeName: Unconventional myosin-Va with calmodulin-1 bound / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #4: Calmodulin-1

SupramoleculeName: Calmodulin-1 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #1 / Details: 1 calmodulin bound per IQ domain (6 overall)
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Calmodulin-1

MacromoleculeName: Calmodulin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 16.72135 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
ADQLTEEQIA EFKEAFSLFD KDGDGTITTK ELGTVMRSLG QNPTEAELQD MINEVDADGN GTIDFPEFLT MMARKMKDTD SEEEIREAF RVFDKDGNGY ISAAELRHVM TNLGEKLTDE EVDEMIREAD IDGDGQVNYE EFVQMMTAK

UniProtKB: Calmodulin-1

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Macromolecule #2: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 2 / Details: HIC is tele-methylhistidine / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle
Molecular weightTheoretical: 41.875633 KDa
SequenceString: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG ...String:
DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #3: Unconventional myosin-Va

MacromoleculeName: Unconventional myosin-Va / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 106.596195 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAASELYTKF ARVWIPDPEE VWKSAELLKD YKPGDKVLLL HLEEGKDLEY RLDPKTGELP HLRNPDILVG ENDLTALSYL HEPAVLHNL RVRFIDSKLI YTYCGIVLVA INPYEQLPIY GEDIINAYSG QNMGDMDPHI FAVAEEAYKQ MARDERNQSI I VSGESGAG ...String:
MAASELYTKF ARVWIPDPEE VWKSAELLKD YKPGDKVLLL HLEEGKDLEY RLDPKTGELP HLRNPDILVG ENDLTALSYL HEPAVLHNL RVRFIDSKLI YTYCGIVLVA INPYEQLPIY GEDIINAYSG QNMGDMDPHI FAVAEEAYKQ MARDERNQSI I VSGESGAG KTVSAKYAMR YFATVSGSAS EANVEEKVLA SNPIMESIGN AKTTRNDNSS RFGKYIEIGF DKRYRIIGAN MR TYLLEKS RVVFQAEEER NYHIFYQLCA SAKLPEFKML RLGNADSFHY TKQGGSPMIE GVDDAKEMAH TRQACTLLGI SES YQMGIF RILAGILHLG NVGFASRDSD SCTIPPKHEP LTIFCDLMGV DYEEMCHWLC HRKLATATET YIKPISKLQA TNAR DALAK HIYAKLFNWI VDHVNQALHS AVKQHSFIGV LDIYGFETFE INSFEQFCIN YANEKLQQQF NMHVFKLEQE EYMKE QIPW TLIDFYDNQP CINLIESKLG ILDLLDEECK MPKGTDDTWA QKLYNTHLNK CALFEKPRMS NKAFIIKHFA DKVEYQ CEG FLEKNKDTVF EEQIKVLKSS KFKMLPELFQ DDEKAISPTS ATSSGRTPLT RVPVKPTKGR PGQTAKEHKK TVGHQFR NS LHLLMETLNA TTPHYVRCIK PNDFKFPFTF DEKRAVQQLR ACGVLETIRI SAAGFPSRWT YQEFFSRYRV LMKQKDVL G DRKQTCKNVL EKLILDKDKY QFGKTKIFFR AGQVAYLEKL RADKLRAACI RIQKTIRGWL LRKRYLCMQR AAITVQRYV RGYQARCYAK FLRRTKAATT IQKYWRMYVV RRRYKIRRAA TIVIQSYLRG YLTRNRYRKI LREYKAVIIQ KRVRGWLART HYKRTMKAI VYLQCCFRRM MAKRELKKDY KDDDDK

UniProtKB: Unconventional myosin-Va

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7
Component:
ConcentrationFormulaName
100.0 mMKClpotassium chloride
0.1 mMC14H24N2O10egtazic acid (EGTA)
1.0 mMMgCl2magnesium chloride
10.0 mMC7H15NO4S3-morpholinopropane-1-sulfonic acid (MOPS)
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AMYLAMINE
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV
Details5.26 mg/mL Actin, alpha skeletal muscle 266 mg/mL FlAG-tagged unconventional myosin-Va (residues 1-907) 84.2 mg/mL Calmodulin-1

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 4285 / Average exposure time: 1.5 sec. / Average electron dose: 63.13 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 838213
Startup modelType of model: OTHER / Details: relion_reconstruct map from particles in 3D class
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 22337
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 20 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

7plu

chain_id: D, source_name: SwissModel, initial_model_type: in silico modelchain D was used as a template to model the motor domain
source_name: AlphaFold, initial_model_type: in silico modelthe lever structure with CaM bound was predicted in AlphaFold

7plu

source_name: PDB, initial_model_type: experimental modelchains J-L were used to model actin
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8of8:
Cryo-EM structure of actomyosin-5a-S1 with the full-length lever (nucleotide free)

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