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- PDB-9fju: Structure of the DNase I- and phalloidin-bound pointed end of F-a... -

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Basic information

Entry
Database: PDB / ID: 9fju
TitleStructure of the DNase I- and phalloidin-bound pointed end of F-actin (conformer 1)
Components
  • Actin, cytoplasmic 1, N-terminally processed
  • Deoxyribonuclease-1
  • Phalloidin
KeywordsSTRUCTURAL PROTEIN / actin / phalloidin / filament / pointed end / DNase I
Function / homology
Function and homology information


Cell-extracellular matrix interactions / Formation of the dystrophin-glycoprotein complex (DGC) / Adherens junctions interactions / regulation of neutrophil mediated cytotoxicity / B-WICH complex positively regulates rRNA expression / zymogen granule / regulation of acute inflammatory response / Gap junction degradation / Formation of annular gap junctions / MAP2K and MAPK activation ...Cell-extracellular matrix interactions / Formation of the dystrophin-glycoprotein complex (DGC) / Adherens junctions interactions / regulation of neutrophil mediated cytotoxicity / B-WICH complex positively regulates rRNA expression / zymogen granule / regulation of acute inflammatory response / 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 / deoxyribonuclease I activity / VEGFA-VEGFR2 Pathway / neutrophil activation involved in immune response / structural constituent of postsynaptic actin cytoskeleton / Clathrin-mediated endocytosis / dense body / DNA catabolic process / NuA4 histone acetyltransferase complex / axonogenesis / actin filament / cell motility / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / nuclear envelope / actin cytoskeleton / actin binding / cytoskeleton / hydrolase activity / axon / focal adhesion / apoptotic process / synapse / protein kinase binding / protein-containing complex / DNA binding / extracellular region / ATP binding / nucleus / membrane / 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
: / ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Deoxyribonuclease-1 / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesBos taurus (domestic cattle)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsBoiero Sanders, M. / Oosterheert, W. / Hofnagel, O. / Bieling, P. / Raunser, S.
Funding support Germany, European Union, 3items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 2, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actin, cytoplasmic 1, N-terminally processed
B: Actin, cytoplasmic 1, N-terminally processed
C: Actin, cytoplasmic 1, N-terminally processed
D: Actin, cytoplasmic 1, N-terminally processed
E: Deoxyribonuclease-1
F: Deoxyribonuclease-1
G: Phalloidin
H: Phalloidin
I: Phalloidin
J: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)235,77428
Polymers232,64210
Non-polymers3,13218
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 6 molecules ABCDEF

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


Mass: 41664.484 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Details: Beta/gamma actin mix purified from bovine thymus. / Source: (natural) Bos taurus (domestic cattle) / Organ: Thymus / References: UniProt: P60712
#2: Protein Deoxyribonuclease-1 / Deoxyribonuclease I / DNase I


Mass: 31374.436 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Details: DNase I from bovine pancreas (DNase I, Serva, cat. no. 18535.02)
Source: (natural) Bos taurus (domestic cattle) / Organ: Pancreas / References: UniProt: P00639, deoxyribonuclease I

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Protein/peptide / Sugars , 2 types, 6 molecules GHIJ

#3: Protein/peptide
Phalloidin


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Amanita phalloides (death cap) / References: BIRD: PRD_002366
#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE

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Non-polymers , 3 types, 16 molecules

#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#7: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1F-actin pointed end bound by DNase I and phalloidin.COMPLEXBovine 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.#1-#30MULTIPLE SOURCES
2Actin filament pointed endCOMPLEXThe four terminal subunits of the pointed end of the actin filament.#11NATURAL
3PhalloidinCOMPLEXToxin from Amanita phalloides that stabilizes the actin filament.#31NATURAL
4Two DNase I molecules, each bound to the ultimate and penultimate actin subunits of the F-actin pointed end.COMPLEXBovine DNase I was bought from Serva.#21NATURAL
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22NO
33NO
44NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDOrgan
22Bos taurus (domestic cattle)9913Thymus
33Amanita phalloides (death cap)67723
44Bos taurus (domestic cattle)9913Pancreas
Buffer solutionpH: 7 / Details: 1xKMEI plus phalloidin
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMImidazole1
250 mMpotassium chlorideKCl1
31.5 mMmagnesium chlorideMgCl21
41 mMEGTA1
51 mMTCEP1
60.5 mMATP1
745.5 uMPhalloidin1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 286 K / Details: 3 seconds, force 0.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: 300 kV Titan Krios G2 microscope (Thermo Fisher Scientific) with an in-column Cs-corrector.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 0.01 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 15978
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: Gatan energy filter. / Energyfilter slit width: 15 eV
Spherical aberration corrector: Titan Krios G2 microscope (Thermo Fisher Scientific) with an in-column Cs-corrector.

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Processing

EM software
IDNameVersionCategory
1crYOLO1.8particle selection
2EPUimage acquisition
4CTFFIND4.13CTF correction
7UCSF ChimeraX1.6.1model fitting
8Coot0.9.8.1model fitting
10cryoSPARCv3.3.2initial Euler assignment
11cryoSPARCv4.2.1final Euler assignment
12cryoSPARCv4.2.1classification
13cryoSPARCv4.2.13D reconstruction
14Coot0.9.8.1model refinement
15PHENIX1.21rc1_5015model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4418492 / Details: Particles picked using SPHIRE-crYOLO.
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161657 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: Real Space Refinement in Phenix.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeDetailsInitial refinement model-ID
16T20

6t20

6T20Actin and phalloidin model1
22A42

2a42

2A42DNase I model2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00216314
ELECTRON MICROSCOPYf_angle_d0.56422170
ELECTRON MICROSCOPYf_dihedral_angle_d6.7712250
ELECTRON MICROSCOPYf_chiral_restr0.0442498
ELECTRON MICROSCOPYf_plane_restr0.0032806

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