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- PDB-8ru2: Structure of the F-actin barbed end bound by formin mDia1 -

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Basic information

Entry
Database: PDB / ID: 8ru2
TitleStructure of the F-actin barbed end bound by formin mDia1
Components
  • Actin, cytoplasmic 1, N-terminally processed
  • Methylated-DNA--protein-cysteine methyltransferase,Protein diaphanous homolog 1
KeywordsSTRUCTURAL PROTEIN / actin / formin / Cdc12 / profilin / actin assembly
Function / homology
Function and homology information


negative regulation of neuron projection regeneration / multicellular organismal locomotion / MGMT-mediated DNA damage reversal / RHOF GTPase cycle / RHOB GTPase cycle / ERBB2 Regulates Cell Motility / RHOC GTPase cycle / RHOD GTPase cycle / RHOA GTPase cycle / methylated-DNA-[protein]-cysteine S-methyltransferase ...negative regulation of neuron projection regeneration / multicellular organismal locomotion / MGMT-mediated DNA damage reversal / RHOF GTPase cycle / RHOB GTPase cycle / ERBB2 Regulates Cell Motility / RHOC GTPase cycle / RHOD GTPase cycle / RHOA GTPase cycle / methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / actin nucleation / positive regulation of norepinephrine uptake / neuron projection retraction / cellular response to cytochalasin B / DNA-methyltransferase activity / bBAF complex / RHO GTPases Activate Formins / npBAF complex / protein localization to microtubule / postsynaptic actin cytoskeleton organization / regulation of transepithelial transport / brahma complex / nBAF complex / structural constituent of postsynaptic actin cytoskeleton / morphogenesis of a polarized epithelium / profilin binding / cellular response to histamine / Formation of annular gap junctions / GBAF complex / Gap junction degradation / postsynaptic actin cytoskeleton / protein localization to adherens junction / regulation of G0 to G1 transition / dense body / Cell-extracellular matrix interactions / Tat protein binding / DNA alkylation repair / Folding of actin by CCT/TriC / regulation of double-strand break repair / DNA ligation / regulation of nucleotide-excision repair / RSC-type complex / apical protein localization / Prefoldin mediated transfer of substrate to CCT/TriC / regulation of microtubule-based process / regulation of release of sequestered calcium ion into cytosol / adherens junction assembly / RHOF GTPase cycle / Adherens junctions interactions / axon midline choice point recognition / tight junction / Sensory processing of sound by outer hair cells of the cochlea / SWI/SNF complex / Interaction between L1 and Ankyrins / Sensory processing of sound by inner hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / regulation of norepinephrine uptake / positive regulation of double-strand break repair / positive regulation of T cell differentiation / NuA4 histone acetyltransferase complex / regulation of synaptic vesicle endocytosis / apical junction complex / regulation of cytoskeleton organization / maintenance of blood-brain barrier / establishment or maintenance of cell polarity / cortical cytoskeleton / positive regulation of double-strand break repair via homologous recombination / positive regulation of stem cell population maintenance / nitric-oxide synthase binding / Recycling pathway of L1 / regulation of cyclin-dependent protein serine/threonine kinase activity / regulation of G1/S transition of mitotic cell cycle / negative regulation of cell differentiation / brush border / kinesin binding / calyx of Held / synaptic vesicle endocytosis / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / ephrin receptor signaling pathway / positive regulation of myoblast differentiation / regulation of protein localization to plasma membrane / cytoskeleton organization / EPHB-mediated forward signaling / substantia nigra development / actin filament polymerization / Neutrophil degranulation / axonogenesis / negative regulation of protein binding / actin filament / cell motility / methyltransferase activity / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of transmembrane transporter activity / positive regulation of cell differentiation / sensory perception of sound / FCGR3A-mediated phagocytosis
Similarity search - Function
Formin Homology Region 1 / Diaphanous, GTPase-binding domain superfamily / Diaphanous autoregulatory (DAD) domain / Diaphanous autoregulatory domain (DAD) profile. / Formin, FH3 domain / Formin, GTPase-binding domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain ...Formin Homology Region 1 / Diaphanous, GTPase-binding domain superfamily / Diaphanous autoregulatory (DAD) domain / Diaphanous autoregulatory domain (DAD) profile. / Formin, FH3 domain / Formin, GTPase-binding domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain profile. / Methylguanine DNA methyltransferase, ribonuclease-like domain / 6-O-methylguanine DNA methyltransferase, ribonuclease-like domain / Formin, FH2 domain / Formin, FH2 domain superfamily / Formin Homology 2 Domain / Formin homology-2 (FH2) domain profile. / Formin Homology 2 Domain / Methylated DNA-protein cysteine methyltransferase domain superfamily / Methylated-DNA-[protein]-cysteine S-methyltransferase, active site / Methylated-DNA--protein-cysteine methyltransferase active site. / Methylated-DNA-[protein]-cysteine S-methyltransferase, DNA binding / Methylated DNA-protein cysteine methyltransferase, DNA binding domain / 6-O-methylguanine DNA methyltransferase, DNA binding domain / 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 / Armadillo-like helical / Armadillo-type fold / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Protein diaphanous homolog 1 / Methylated-DNA--protein-cysteine methyltransferase / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsOosterheert, W. / Boiero Sanders, M. / Funk, J. / Prumbaum, D. / Raunser, S. / Bieling, P.
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: Science / Year: 2024
Title: Molecular mechanism of actin filament elongation by formins.
Authors: Wout Oosterheert / Micaela Boiero Sanders / Johanna Funk / Daniel Prumbaum / Stefan Raunser / Peter Bieling /
Abstract: Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action ...Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.
History
DepositionJan 29, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Actin, cytoplasmic 1, N-terminally processed
C: Actin, cytoplasmic 1, N-terminally processed
D: Actin, cytoplasmic 1, N-terminally processed
E: Methylated-DNA--protein-cysteine methyltransferase,Protein diaphanous homolog 1
F: Methylated-DNA--protein-cysteine methyltransferase,Protein diaphanous homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)299,19011
Polymers297,8365
Non-polymers1,3556
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 41632.422 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Human beta-actin was recombinantly purified from BTI-Tnao38 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 Methylated-DNA--protein-cysteine methyltransferase,Protein diaphanous homolog 1 / 6-O-methylguanine-DNA methyltransferase / MGMT / O-6-methylguanine-DNA-alkyltransferase / ...6-O-methylguanine-DNA methyltransferase / MGMT / O-6-methylguanine-DNA-alkyltransferase / Diaphanous-related formin-1 / DRF1 / p140mDIA / mDIA1


Mass: 86469.258 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Has a N-terminal snap-tag.
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Mus musculus (house mouse)
Gene: MGMT, Diaph1, Diap1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Star pRARE
References: UniProt: P16455, UniProt: O08808, methylated-DNA-[protein]-cysteine S-methyltransferase
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / 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.: 3 / Source method: obtained synthetically / Formula: Mg
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
1mDia1-bound F-actin barbed end.COMPLEXHuman beta-actin and mouse mDia1 were purified separately. Both proteins were mixed to assemble the complex prior to cryo-EM grid preparation.#1-#20MULTIPLE SOURCES
2Actin filamentMicrofilamentCOMPLEX#11RECOMBINANT
3Mouse mDia1 (FH1FH2C domain)COMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Homo sapiens (human)9606
43Mus musculus (house mouse)10090
53Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCell
32Trichoplusia ni (cabbage looper)7111BTI-Tnao38
43Escherichia coli (E. coli)562BL21 Star pRARE
Buffer solutionpH: 7.1
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
270 mMpotassium chlorideKCl1
30.01 % (v/v)Tween201
40.5 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 FIELDBright-field microscopy / 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: 67.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 38913
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
10cryoSPARCv4.3.0initial Euler assignment
11cryoSPARCv4.3.0final Euler assignment
12cryoSPARCv4.3.0classification
13cryoSPARCv4.3.03D reconstruction
14Coot0.9.8.1model refinement
15PHENIX1.21rc1_5015model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1963686 / Details: Particles picked using SPHIRE-crYOLO.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150807 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Refinement performed using phenix real-space refine
Atomic model building

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

IDPDB-IDAccession codeDetailsInitial refinement model-ID
18RTT

8rtt

8RTTActin model1
23OBV

3obv

3OBVmDia1 model2
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 115.26 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002415068
ELECTRON MICROSCOPYf_angle_d0.531520351
ELECTRON MICROSCOPYf_chiral_restr0.03942247
ELECTRON MICROSCOPYf_plane_restr0.00342617
ELECTRON MICROSCOPYf_dihedral_angle_d5.8892025

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