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

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Basic information

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


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 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: 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 MSC / Klebl DP / Harlen OG / Read DJ / Harris SA / Muench SP / Peckham M
History
DepositionMar 14, 2023-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing 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
Contrast
Others
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

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

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
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: Gaussian filtered (SD 5) map

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

Fileemd_16850_additional_2.map
AnnotationMasked post-processed map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw map

Fileemd_16850_additional_3.map
AnnotationRaw map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_16850_half_map_1.map
AnnotationHalf map 1
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_16850_half_map_2.map
AnnotationHalf map 2
Projections & Slices
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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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