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- EMDB-50517: Structure of the DNase I- and phalloidin-bound pointed end of F-a... -

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Basic information

Entry
Database: EMDB / ID: EMD-50517
TitleStructure of the DNase I- and phalloidin-bound pointed end of F-actin (conformer 2).
Map dataSharpened cryo-EM density map of the DNase I- and phalloidin-bound pointed end of actin filaments (conformer 2).
Sample
  • Complex: F-actin pointed end bound by DNase I and phalloidin.
    • Complex: Actin filament pointed end
      • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
    • Complex: Phalloidin
      • Protein or peptide: Phalloidin
    • Complex: Two DNase I molecules, each bound to the ultimate and penultimate actin subunits of the F-actin pointed end.
      • Protein or peptide: Deoxyribonuclease-1
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE ION
Keywordsactin / phalloidin / filament / pointed end / DNase I / STRUCTURAL PROTEIN
Function / homology
Function and homology information


Cell-extracellular matrix interactions / Adherens junctions interactions / B-WICH complex positively regulates rRNA expression / regulation of neutrophil mediated cytotoxicity / regulation of acute inflammatory response / zymogen granule / Gap junction degradation / Formation of annular gap junctions / MAP2K and MAPK activation / RHOF GTPase cycle ...Cell-extracellular matrix interactions / Adherens junctions interactions / B-WICH complex positively regulates rRNA expression / regulation of neutrophil mediated cytotoxicity / regulation of acute inflammatory response / zymogen granule / 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 / deoxyribonuclease I / DNA Damage Recognition in GG-NER / UCH proteinases / VEGFA-VEGFR2 Pathway / deoxyribonuclease I activity / neutrophil activation involved in immune response / structural constituent of postsynaptic actin cytoskeleton / dense body / Clathrin-mediated endocytosis / DNA catabolic process / NuA4 histone acetyltransferase complex / axonogenesis / cell motility / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / actin cytoskeleton / nuclear envelope / actin binding / cytoskeleton / hydrolase activity / axon / focal adhesion / synapse / apoptotic process / protein kinase binding / protein-containing complex / DNA binding / extracellular region / ATP binding / membrane / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Deoxyribonuclease I / Deoxyribonuclease I, active site / Deoxyribonuclease I, conservied site / Deoxyribonuclease I signature 2. / Deoxyribonuclease I signature 1. / deoxyribonuclease I / Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase superfamily / Actins signature 1. ...Deoxyribonuclease I / Deoxyribonuclease I, active site / Deoxyribonuclease I, conservied site / Deoxyribonuclease I signature 2. / Deoxyribonuclease I signature 1. / deoxyribonuclease I / Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase 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 / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Deoxyribonuclease-1 / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesBos taurus (cattle) / Amanita phalloides (death cap)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.79 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_50517.png

Downloads & links

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Map

FileDownload / File: emd_50517.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM density map of the DNase I- and phalloidin-bound pointed end of actin filaments (conformer 2).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 320 pix.
= 281.6 Å		0.88 Å/pix.
x 320 pix.
= 281.6 Å		0.88 Å/pix.
x 320 pix.
= 281.6 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: 0.343
Minimum - Maximum-0.78308713 - 1.6706626
Average (Standard dev.)0.0030969903 (±0.046890896)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50517_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 DNase I-...

Fileemd_50517_additional_1.map
AnnotationUnsharpened cryo-EM density map of the DNase I- and phalloidin-bound pointed end of actin filaments (conformer 2).
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 DNase I-...

Fileemd_50517_half_map_1.map
AnnotationUnfiltered half map 1 of the DNase I- and phalloidin-bound pointed end of actin filaments (conformer 2).
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 DNase I-...

Fileemd_50517_half_map_2.map
AnnotationUnfiltered half map 2 of the DNase I- and phalloidin-bound pointed end of actin filaments (conformer 2).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : F-actin pointed end bound by DNase I and phalloidin.

EntireName: F-actin pointed end bound by DNase I and phalloidin.
Components
  • Complex: F-actin pointed end bound by DNase I and phalloidin.
    • Complex: Actin filament pointed end
      • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
    • Complex: Phalloidin
      • Protein or peptide: Phalloidin
    • Complex: Two DNase I molecules, each bound to the ultimate and penultimate actin subunits of the F-actin pointed end.
      • Protein or peptide: Deoxyribonuclease-1
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE ION

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Supramolecule #1: F-actin pointed end bound by DNase I and phalloidin.

SupramoleculeName: F-actin pointed end bound by DNase I and phalloidin. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Bovine beta/gamma-actin was purified from bovine thymus, phalloidin (from Amanita phalloides) was bought from Sigma, DNase I was bought from Serva. The components were mixed to assemble the ...Details: Bovine beta/gamma-actin was purified from bovine thymus, phalloidin (from Amanita phalloides) was bought from Sigma, DNase I was bought from Serva. 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: Bos taurus (cattle) / Organ: Thymus

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Supramolecule #3: Phalloidin

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

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Supramolecule #4: Two DNase I molecules, each bound to the ultimate and penultimate...

SupramoleculeName: Two DNase I molecules, each bound to the ultimate and penultimate actin subunits of the F-actin pointed end.
type: complex / ID: 4 / Parent: 1 / Macromolecule list: #2 / Details: Bovine DNase I was bought from Serva.
Source (natural)Organism: Bos taurus (cattle) / Organ: Pancreas

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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/gamma actin mix purified from bovine thymus. / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle) / Organ: Thymus
Molecular weightTheoretical: 41.664484 KDa
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 MESCGIHETT FNSIMKCDVD IRKDLYANTV LSGGTTMYPG IADRMQKEI TALAPSTMKI KIIAPPERKY SVWIGGSILA SLSTFQQMWI SKQEYDESGP SIVHRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #2: Deoxyribonuclease-1

MacromoleculeName: Deoxyribonuclease-1 / type: protein_or_peptide / ID: 2
Details: DNase I from bovine pancreas (DNase I, Serva, cat. no 18535.02)
Number of copies: 2 / Enantiomer: LEVO / EC number: deoxyribonuclease I
Source (natural)Organism: Bos taurus (cattle) / Organ: Pancreas
Molecular weightTheoretical: 31.374436 KDa
SequenceString: MRGTRLMGLL LALAGLLQLG LSLKIAAFNI RTFGETKMSN ATLASYIVRI VRRYDIVLIQ EVRDSHLVAV GKLLDYLNQD DPNTYHYVV SEPLGRNSYK ERYLFLFRPN KVSVLDTYQY DDGCESCGND SFSREPAVVK FSSHSTKVKE FAIVALHSAP S DAVAEINS ...String:
MRGTRLMGLL LALAGLLQLG LSLKIAAFNI RTFGETKMSN ATLASYIVRI VRRYDIVLIQ EVRDSHLVAV GKLLDYLNQD DPNTYHYVV SEPLGRNSYK ERYLFLFRPN KVSVLDTYQY DDGCESCGND SFSREPAVVK FSSHSTKVKE FAIVALHSAP S DAVAEINS LYDVYLDVQQ KWHLNDVMLM GDFNADCSYV TSSQWSSIRL RTSSTFQWLI PDSADTTATS TNCAYDRIVV AG SLLQSSV VPGSAAPFDF QAAYGLSNEM ALAISDHYPV EVTLT

UniProtKB: Deoxyribonuclease-1

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Macromolecule #3: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 3 / 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 #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / 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 #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 7 / 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
Component:
ConcentrationNameFormula
10.0 mMImidazole
50.0 mMpotassium chlorideKCl
1.5 mMmagnesium chlorideMgCl2
1.0 mMEGTA
1.0 mMTCEP
0.5 mMATP
45.5 uMPhalloidin

Details: 1xKMEI plus phalloidin
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: 2 / Number real images: 15978 / 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: 2.5 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: 4418492 / Details: Particles picked using SPHIRE-crYOLO.
Startup modelType of model: OTHER
Details: Low resolution map of the pointed end bound by DNase I generated from a previous dataset.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.2.1) / Number images used: 178346
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.2.1)
Final 3D classificationNumber classes: 4 / 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 model
PDB IDChainDetails

6t20

source_name: PDB, initial_model_type: experimental modelActin and phalloidin model

2a42

source_name: PDB, initial_model_type: experimental modelDNase I model
DetailsReal Space Refinement in Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9fjy:
Structure of the DNase I- and phalloidin-bound pointed end of F-actin (conformer 2).

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