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

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Basic information

Entry
Database: PDB / ID: 9fjm
TitleCryo-EM structure of the phalloidin-bound pointed end of the actin filament.
Components
  • Actin, cytoplasmic 1, N-terminally processed
  • Phalloidin
KeywordsSTRUCTURAL PROTEIN / actin / phalloidin / filament / pointed end
Function / homology
Function and homology information


positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / regulation of transepithelial transport / nBAF complex / brahma complex / morphogenesis of a polarized epithelium / protein localization to adherens junction / postsynaptic actin cytoskeleton ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / regulation of transepithelial transport / nBAF complex / brahma complex / morphogenesis of a polarized epithelium / protein localization to adherens junction / postsynaptic actin cytoskeleton / structural constituent of postsynaptic actin cytoskeleton / Formation of the dystrophin-glycoprotein complex (DGC) / GBAF complex / Formation of annular gap junctions / Tat protein binding / regulation of G0 to G1 transition / Gap junction degradation / Folding of actin by CCT/TriC / Cell-extracellular matrix interactions / dense body / regulation of nucleotide-excision repair / regulation of double-strand break repair / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / apical protein localization / adherens junction assembly / RHOF GTPase cycle / Adherens junctions interactions / tight junction / Sensory processing of sound by outer hair cells of the cochlea / Interaction between L1 and Ankyrins / SWI/SNF complex / regulation of mitotic metaphase/anaphase transition / Sensory processing of sound by inner hair cells of the cochlea / positive regulation of T cell differentiation / regulation of norepinephrine uptake / transporter regulator activity / apical junction complex / nitric-oxide synthase binding / positive regulation of double-strand break repair / maintenance of blood-brain barrier / NuA4 histone acetyltransferase complex / establishment or maintenance of cell polarity / cortical cytoskeleton / positive regulation of stem cell population maintenance / Regulation of MITF-M-dependent genes involved in pigmentation / regulation of synaptic vesicle endocytosis / Recycling pathway of L1 / regulation of G1/S transition of mitotic cell cycle / brush border / kinesin binding / negative regulation of cell differentiation / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / positive regulation of myoblast differentiation / RHO GTPases activate IQGAPs / positive regulation of double-strand break repair via homologous recombination / regulation of protein localization to plasma membrane / cytoskeleton organization / EPHB-mediated forward signaling / substantia nigra development / calyx of Held / axonogenesis / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / adherens junction / positive regulation of cell differentiation / actin filament / FCGR3A-mediated phagocytosis / cell motility / RHO GTPases Activate Formins / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / DNA Damage Recognition in GG-NER / Schaffer collateral - CA1 synapse / B-WICH complex positively regulates rRNA expression / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / kinetochore / Regulation of actin dynamics for phagocytic cup formation / structural constituent of cytoskeleton / tau protein binding / VEGFA-VEGFR2 Pathway / platelet aggregation / cytoplasmic ribonucleoprotein granule / nuclear matrix / 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 / Signaling by BRAF and RAF1 fusions / nucleosome / cell-cell junction / lamellipodium / actin cytoskeleton / presynapse / 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
: / ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 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

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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
H: Phalloidin
I: Phalloidin
K: Phalloidin
J: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,95120
Polymers169,7658
Non-polymers2,18612
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

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


Mass: 41632.422 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Beta-actin recombinantly purified from insect cells.
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTB / Plasmid: p2336 pFL_ACTB_C272A / Cell line (production host): BTI-Tnao38 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P60709
#2: Protein/peptide
Phalloidin


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Details: phalloidin from Amanita phalloides. / Source: (natural) Amanita phalloides (death cap) / References: BIRD: PRD_002366
#3: 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
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
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
1Actin filament pointed end bound by phalloidinCOMPLEXHuman 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.#1-#20MULTIPLE SOURCES
2Actin filament pointed endCOMPLEXThe four terminal subunits of the pointed end of the actin filament.#11RECOMBINANT
3PhalloidinCOMPLEXToxin from Amanita phalloides that stabilizes the actin filament#21NATURAL
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22NO
33NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Amanita phalloides (death cap)67723
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Cell: BTI-Tnao38
Buffer solutionpH: 7.1
Details: 1xKMEH (10 mM HEPES pH 7.1, 100 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.5 mM TCEP)
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
2100 mMpotassium chlorideKCl1
32 mMmagnesium chlorideMgCl21
41 mMEGTA1
50.5 mMTCEP1
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: 2700 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: 1 / Num. of real images: 20393
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
1crYOLO1.5.8particle selection
2EPUimage acquisition
4CTFFIND4.13CTF correction
7UCSF ChimeraX1.5model fitting
8Coot0.9.8.1model fitting
10cryoSPARCv3.3.2initial Euler assignment
11cryoSPARCv4.0.3final 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: 5200600 / Details: Particles picked using SPHIRE-crYOLO.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 280802 / Details: Local Refinement in CryoSPARC. / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 8rtt

8rtt


Accession code: 8rtt / Details: actin and phalloidin model / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212028
ELECTRON MICROSCOPYf_angle_d0.55216332
ELECTRON MICROSCOPYf_dihedral_angle_d7.3681672
ELECTRON MICROSCOPYf_chiral_restr0.0421816
ELECTRON MICROSCOPYf_plane_restr0.0042068

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