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- EMDB-16887: Cryo-EM structure of the undecorated barbed end of filamentous be... -

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Basic information

Entry
Database: EMDB / ID: EMD-16887
TitleCryo-EM structure of the undecorated barbed end of filamentous beta/gamma actin
Map data3D-refined, sharpened cryo-EM density map of the undecorated barbed end of F-actin.
Sample
  • Complex: actin-phalloidin complex
    • Complex: cytosolic beta-gamma actin
    • Complex: phalloidin
    • Protein or peptide: Actin, cytoplasmic 1
  • Protein or peptide: PHALLOIDIN
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsActin filament / cytoskeletal protein / ATPase / STRUCTURAL PROTEIN
Function / homology
Function and homology information


Cell-extracellular matrix interactions / Adherens junctions interactions / Formation of the dystrophin-glycoprotein complex (DGC) / B-WICH complex positively regulates rRNA expression / Gap junction degradation / Formation of annular gap junctions / MAP2K and MAPK activation / RHOF GTPase cycle / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation ...Cell-extracellular matrix interactions / Adherens junctions interactions / Formation of the dystrophin-glycoprotein complex (DGC) / B-WICH complex positively regulates rRNA expression / Gap junction degradation / Formation of annular gap junctions / MAP2K and MAPK activation / RHOF GTPase cycle / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / DNA Damage Recognition in GG-NER / UCH proteinases / VEGFA-VEGFR2 Pathway / cellular response to cytochalasin B / regulation of transepithelial transport / morphogenesis of a polarized epithelium / structural constituent of postsynaptic actin cytoskeleton / Clathrin-mediated endocytosis / protein localization to adherens junction / dense body / postsynaptic actin cytoskeleton / Tat protein binding / adherens junction assembly / apical protein localization / tight junction / apical junction complex / regulation of norepinephrine uptake / transporter regulator activity / nitric-oxide synthase binding / cortical cytoskeleton / establishment or maintenance of cell polarity / NuA4 histone acetyltransferase complex / brush border / kinesin binding / regulation of synaptic vesicle endocytosis / regulation of protein localization to plasma membrane / positive regulation of double-strand break repair via homologous recombination / axonogenesis / calyx of Held / nitric-oxide synthase regulator activity / adherens junction / actin filament / cell motility / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Schaffer collateral - CA1 synapse / cytoplasmic ribonucleoprotein granule / nucleosome / lamellipodium / actin cytoskeleton / cytoskeleton / regulation of cell cycle / ribonucleoprotein complex / axon / focal adhesion / synapse / protein kinase binding / glutamatergic synapse / protein-containing complex / ATP hydrolysis activity / ATP binding / identical protein binding / nucleus / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Actin, cytoplasmic 1
Similarity search - Component
Biological speciesBos taurus (domestic cattle) / Amanita phalloides (death cap)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsOosterheert W / Blanc FEC / Roy A / Belyy A / Hofnagel O / Hummer G / Bieling P / Raunser S
Funding support Germany, European Union, 3 items
OrganizationGrant numberCountry
Max Planck Society Germany
Alexander von Humboldt Foundation Germany
European Research Council (ERC)856118European Union
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments.
Authors: Wout Oosterheert / Florian E C Blanc / Ankit Roy / Alexander Belyy / Micaela Boiero Sanders / Oliver Hofnagel / Gerhard Hummer / Peter Bieling / Stefan Raunser /
Abstract: The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release ...The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases P through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why P escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated P release and filaments with drastically shortened ADP-P caps. Our results provide the molecular basis for P release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament.
History
DepositionMar 22, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16887.png

Downloads & links

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Map

FileDownload / File: emd_16887.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D-refined, sharpened cryo-EM density map of the undecorated barbed end of F-actin.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.21 Å/pix.
x 384 pix.
= 464.64 Å		1.21 Å/pix.
x 384 pix.
= 464.64 Å		1.21 Å/pix.
x 384 pix.
= 464.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.25
Minimum - Maximum-3.310238 - 9.226526
Average (Standard dev.)-0.0017824335 (±0.21388076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 464.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16887_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D-refined, unsharpened cryo-EM density map of the undecorated...

Fileemd_16887_additional_1.map
Annotation3D-refined, unsharpened cryo-EM density map of the undecorated barbed end of F-actin.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of the refinement of the...

Fileemd_16887_half_map_1.map
AnnotationHalf map 2 of the refinement of the undecorated barbed end of F-actin.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of the refinement of the...

Fileemd_16887_half_map_2.map
AnnotationHalf map 1 of the refinement of the undecorated barbed end of F-actin.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : actin-phalloidin complex

EntireName: actin-phalloidin complex
Components
  • Complex: actin-phalloidin complex
    • Complex: cytosolic beta-gamma actin
    • Complex: phalloidin
    • Protein or peptide: Actin, cytoplasmic 1
  • Protein or peptide: PHALLOIDIN
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: actin-phalloidin complex

SupramoleculeName: actin-phalloidin complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Actin was purified as monomer from bovine thymus. Short filaments were reconstituted in vitro to obtain the barbed end structure.

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Supramolecule #2: cytosolic beta-gamma actin

SupramoleculeName: cytosolic beta-gamma actin / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Bos taurus (domestic cattle) / Tissue: thymus / Location in cell: cytoplasm

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Supramolecule #3: phalloidin

SupramoleculeName: phalloidin / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Amanita phalloides (death cap)

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Macromolecule #1: Actin, cytoplasmic 1

MacromoleculeName: Actin, cytoplasmic 1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle) / Tissue: thymus
Molecular weightTheoretical: 41.79568 KDa
SequenceString: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG ...String:
MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG YALPHAILRL DLAGRDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PEALFQPSFL GMESCGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #2: PHALLOIDIN

MacromoleculeName: PHALLOIDIN / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Amanita phalloides (death cap)
Molecular weightTheoretical: 808.899 Da
SequenceString:
W(EEP)A(DTH)C(HYP)A

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.1
Component:
ConcentrationNameFormula
10.0 mMimidazole
100.0 mMpotassium chlorideKCl
2.0 mMmagnesium clorideMgCl2
1.0 mMethylene glycol-bis(beta-aminoethyl ether)-N,N,N,N-tetraacetic acidEGTA

Details: 10 mM imidazole pH 7.1, 100 mM KCl, 2 mM MgCl2 and 1 mM EGTA
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was directly collected from size-exclusion chromatography fractions.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
DetailsMicrosope alignment was performed using SHERPA (Thermo Fisher)
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 1316 / Average exposure time: 4.0 sec. / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 120000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsFalcon III operated in linear mode.
Particle selectionNumber selected: 252982 / Details: CrYOLO was trained to pick filament ends.
Startup modelType of model: EMDB MAP
EMDB ID:

3835

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2)
Details: Performed in CryoSPARC. All barbed end particles were isolated from from other particles that represented pointed end and filament core.
Number images used: 43618
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2) / Details: First refinement performed in CryoSPARC.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Details: Final refinement performed in CryoSPARC. The barbed end structure was refined as a single particle without applying any restraints.
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8a2t


Chain - Chain ID: c / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsReal space refinement in phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8oi6:
Cryo-EM structure of the undecorated barbed end of filamentous beta/gamma actin

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