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- EMDB-50506: Cryo-EM structure of the phalloidin-bound pointed end of the acti... -

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Entry
Database: EMDB / ID: EMD-50506
TitleCryo-EM structure of the phalloidin-bound pointed end of the actin filament.
Map dataSharpened cryo-EM density map of the phalloidin-bound pointed end of the actin filament.
Sample
  • Complex: Actin filament pointed end bound by phalloidin
    • Complex: Actin filament pointed end
      • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
    • Complex: Phalloidin
      • Protein or peptide: Phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE ION
Keywordsactin / phalloidin / filament / pointed end / STRUCTURAL PROTEIN
Function / homology
Function and homology information


positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / morphogenesis of a polarized epithelium / bBAF complex / npBAF complex / protein localization to adherens junction / postsynaptic actin cytoskeleton / nBAF complex / brahma complex ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / morphogenesis of a polarized epithelium / bBAF complex / npBAF complex / protein localization to adherens junction / postsynaptic actin cytoskeleton / nBAF complex / brahma complex / Tat protein binding / GBAF complex / Formation of annular gap junctions / structural constituent of postsynaptic actin cytoskeleton / regulation of G0 to G1 transition / Gap junction degradation / dense body / Folding of actin by CCT/TriC / Cell-extracellular matrix interactions / apical protein localization / regulation of double-strand break repair / adherens junction assembly / regulation of nucleotide-excision repair / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / RHOF GTPase cycle / tight junction / Adherens junctions interactions / Sensory processing of sound by outer hair cells of the cochlea / Interaction between L1 and Ankyrins / regulation of mitotic metaphase/anaphase transition / SWI/SNF complex / Sensory processing of sound by inner hair cells of the cochlea / regulation of norepinephrine uptake / positive regulation of double-strand break repair / apical junction complex / positive regulation of T cell differentiation / nitric-oxide synthase binding / regulation of synaptic vesicle endocytosis / establishment or maintenance of cell polarity / regulation of cyclin-dependent protein serine/threonine kinase activity / cortical cytoskeleton / maintenance of blood-brain barrier / NuA4 histone acetyltransferase complex / positive regulation of stem cell population maintenance / Regulation of MITF-M-dependent genes involved in pigmentation / regulation of G1/S transition of mitotic cell cycle / brush border / Recycling pathway of L1 / kinesin binding / negative regulation of cell differentiation / EPH-ephrin mediated repulsion of cells / regulation of protein localization to plasma membrane / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / positive regulation of double-strand break repair via homologous recombination / positive regulation of myoblast differentiation / calyx of Held / substantia nigra development / EPHB-mediated forward signaling / axonogenesis / negative regulation of protein binding / Translocation of SLC2A4 (GLUT4) to the plasma membrane / adherens junction / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / positive regulation of cell differentiation / actin filament / RHO GTPases Activate Formins / FCGR3A-mediated phagocytosis / cell motility / Signaling by high-kinase activity BRAF mutants / regulation of transmembrane transporter activity / DNA Damage Recognition in GG-NER / MAP2K and MAPK activation / tau protein binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Schaffer collateral - CA1 synapse / Regulation of actin dynamics for phagocytic cup formation / B-WICH complex positively regulates rRNA expression / kinetochore / structural constituent of cytoskeleton / platelet aggregation / VEGFA-VEGFR2 Pathway / nuclear matrix / cytoplasmic ribonucleoprotein granule / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / UCH proteinases / cell-cell junction / Signaling by BRAF and RAF1 fusions / nucleosome / actin cytoskeleton / presynapse / lamellipodium / Clathrin-mediated endocytosis / HATs acetylate histones / Factors involved in megakaryocyte development and platelet production / regulation of apoptotic process
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 speciesHomo sapiens (human) / Amanita phalloides (death cap)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsBoiero Sanders M / Oosterheert W / Hofnagel O / Bieling P / Raunser S
Funding support Germany, European Union, 3 items
OrganizationGrant numberCountry
Alexander von Humboldt Foundation Germany
German Research Foundation (DFG)BI 1998/2-1 Germany
European Research Council (ERC)856118European Union
CitationJournal: Nat Commun / Year: 2024
Title: Phalloidin and DNase I-bound F-actin pointed end structures reveal principles of filament stabilization and disassembly.
Authors: Micaela Boiero Sanders / Wout Oosterheert / Oliver Hofnagel / Peter Bieling / Stefan Raunser /
Abstract: Actin filament turnover involves subunits binding to and dissociating from the filament ends, with the pointed end being the primary site of filament disassembly. Several molecules modulate filament ...Actin filament turnover involves subunits binding to and dissociating from the filament ends, with the pointed end being the primary site of filament disassembly. Several molecules modulate filament turnover, but the underlying mechanisms remain incompletely understood. Here, we present three cryo-EM structures of the F-actin pointed end in the presence and absence of phalloidin or DNase I. The two terminal subunits at the undecorated pointed end adopt a twisted conformation. Phalloidin can still bind and bridge these subunits, inducing a conformational shift to a flattened, F-actin-like state. This explains how phalloidin prevents depolymerization at the pointed end. Interestingly, two DNase I molecules simultaneously bind to the phalloidin-stabilized pointed end. In the absence of phalloidin, DNase I binding would disrupt the terminal actin subunit packing, resulting in filament disassembly. Our findings uncover molecular principles of pointed end regulation and provide structural insights into the kinetic asymmetry between the actin filament ends.
History
DepositionMay 31, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50506.png

Downloads & links

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Map

FileDownload / File: emd_50506.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM density map of the phalloidin-bound pointed end of the actin filament.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 480 pix.
= 422.4 Å		0.88 Å/pix.
x 480 pix.
= 422.4 Å		0.88 Å/pix.
x 480 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.648
Minimum - Maximum-1.9884647 - 3.5087998
Average (Standard dev.)-0.0005566647 (±0.051099356)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 422.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50506_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened cryo-EM density map of the phalloidin-bound pointed...

Fileemd_50506_additional_1.map
AnnotationUnsharpened cryo-EM density map of the phalloidin-bound pointed end of the actin filament.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 2 of the phalloidin-bound pointed...

Fileemd_50506_half_map_1.map
AnnotationUnfiltered half map 2 of the phalloidin-bound pointed end of the actin filament.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 1 of the phalloidin-bound pointed...

Fileemd_50506_half_map_2.map
AnnotationUnfiltered half map 1 of the phalloidin-bound pointed end of the actin filament.
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 filament pointed end bound by phalloidin

EntireName: Actin filament pointed end bound by phalloidin
Components
  • Complex: Actin filament pointed end bound by phalloidin
    • Complex: Actin filament pointed end
      • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
    • Complex: Phalloidin
      • Protein or peptide: Phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE ION

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Supramolecule #1: Actin filament pointed end bound by phalloidin

SupramoleculeName: Actin filament pointed end bound by phalloidin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Human beta-actin was purified recombinantly, phalloidin (from Amanita phalloides) was bought from sigma. The components were mixed to assemble the complex prior to cryo-EM grid preparation.

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Supramolecule #2: Actin filament pointed end

SupramoleculeName: Actin filament pointed end / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: The four terminal subunits of the pointed end of the actin filament.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Phalloidin

SupramoleculeName: Phalloidin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: Toxin from Amanita phalloides that stabilizes the actin filament
Source (natural)Organism: Amanita phalloides (death cap)

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Macromolecule #1: Actin, cytoplasmic 1, N-terminally processed

MacromoleculeName: Actin, cytoplasmic 1, N-terminally processed / type: protein_or_peptide / ID: 1
Details: Beta-actin recombinantly purified from insect cells.
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.632422 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: DDDIAALVVD NGSGMCKAGF AGDDAPRAVF PSIVGRPRHQ GVMVGMGQKD SYVGDEAQSK RGILTLKYPI E(HIC)GIVT NWD DMEKIWHHTF YNELRVAPEE HPVLLTEAPL NPKANREKMT QIMFETFNTP AMYVAIQAVL SLYASGRTTG IVMDSGD GV THTVPIYEGY ...String:
DDDIAALVVD NGSGMCKAGF AGDDAPRAVF PSIVGRPRHQ GVMVGMGQKD SYVGDEAQSK RGILTLKYPI E(HIC)GIVT NWD DMEKIWHHTF YNELRVAPEE HPVLLTEAPL NPKANREKMT QIMFETFNTP AMYVAIQAVL SLYASGRTTG IVMDSGD GV THTVPIYEGY ALPHAILRLD LAGRDLTDYL MKILTERGYS FTTTAEREIV RDIKEKLCYV ALDFEQEMAT AASSSSLE K SYELPDGQVI TIGNERFRCP EALFQPSFLG MESAGIHETT FNSIMKCDVD IRKDLYANTV LSGGTTMYPG IADRMQKEI TALAPSTMKI KIIAPPERKY SVWIGGSILA SLSTFQQMWI SKQEYDESGP SIVHRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #2: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 2 / Details: phalloidin from Amanita phalloides. / Number of copies: 4 / 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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Macromolecule #5: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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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 mMHEPES
100.0 mMpotassium chlorideKCl
2.0 mMmagnesium chlorideMgCl2
1.0 mMEGTA
0.5 mMTCEP

Details: 1xKMEH (10 mM HEPES pH 7.1, 100 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.5 mM TCEP)
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / 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 / Details: 3 seconds, force 0..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Titan Krios G2 microscope (Thermo Fisher Scientific) with an in-column Cs-corrector.
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV / Details: Gatan energy filter.
Details300 kV Titan Krios G2 microscope (Thermo Fisher Scientific) with an in-column Cs-corrector.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 20393 / Average electron dose: 64.6 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: 0.01 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
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: 5200600 / Details: Particles picked using SPHIRE-crYOLO.
Startup modelType of model: EMDB MAP
EMDB ID:

15106

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.2.1) / Details: Local Refinement in CryoSPARC. / Number images used: 280802
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.0.3)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. v4.2.1) / Details: 3D classification in CryoSPARC.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8rtt


Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: actin and phalloidin model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9fjm:
Cryo-EM structure of the phalloidin-bound pointed end of the actin filament.

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