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- PDB-8rty: Structure of the F-actin barbed end bound by Cdc12 and profilin (... -

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Basic information

Entry
Database: PDB / ID: 8rty
TitleStructure of the F-actin barbed end bound by Cdc12 and profilin (ring complex) at a resolution of 6.3 Angstrom
Components
  • Actin, cytoplasmic 1, N-terminally processed
  • Methylated-DNA--protein-cysteine methyltransferase,Cell division control protein 12
  • Phalloidin (Amanita phalloides)
  • Profilin-1
KeywordsSTRUCTURAL PROTEIN / actin / formin / Cdc12 / profilin / actin assembly
Function / homology
Function and homology information


F-bar domain binding / protein localization to mitotic actomyosin contractile ring / medial cortical node / mitotic actomyosin contractile ring, proximal layer / mitotic actomyosin contractile ring / MGMT-mediated DNA damage reversal / medial cortex / mitotic actomyosin contractile ring assembly / methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity ...F-bar domain binding / protein localization to mitotic actomyosin contractile ring / medial cortical node / mitotic actomyosin contractile ring, proximal layer / mitotic actomyosin contractile ring / MGMT-mediated DNA damage reversal / medial cortex / mitotic actomyosin contractile ring assembly / methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / positive regulation of norepinephrine uptake / synapse maturation / modification of postsynaptic actin cytoskeleton / negative regulation of actin filament bundle assembly / adenyl-nucleotide exchange factor activity / cellular response to cytochalasin B / DNA-methyltransferase activity / bBAF complex / npBAF complex / postsynaptic actin cytoskeleton organization / regulation of transepithelial transport / brahma complex / nBAF complex / structural constituent of postsynaptic actin cytoskeleton / morphogenesis of a polarized epithelium / positive regulation of actin filament bundle assembly / Formation of annular gap junctions / GBAF complex / Gap junction degradation / postsynaptic actin cytoskeleton / protein localization to adherens junction / negative regulation of actin filament polymerization / regulation of G0 to G1 transition / dense body / Cell-extracellular matrix interactions / Tat protein binding / DNA alkylation repair / Signaling by ROBO receptors / Folding of actin by CCT/TriC / regulation of double-strand break repair / regulation of actin filament polymerization / DNA ligation / regulation of nucleotide-excision repair / RSC-type complex / apical protein localization / mating projection tip / Prefoldin mediated transfer of substrate to CCT/TriC / barbed-end actin filament capping / adherens junction assembly / RHOF GTPase cycle / Adherens junctions interactions / positive regulation of ATP-dependent activity / PCP/CE pathway / proline-rich region binding / tight junction / positive regulation of ruffle assembly / 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 / negative regulation of stress fiber assembly / regulation of synaptic vesicle endocytosis / apical junction complex / maintenance of blood-brain barrier / establishment or maintenance of cell polarity / cell division site / cortical cytoskeleton / positive regulation of double-strand break repair via homologous recombination / positive regulation of actin filament polymerization / positive regulation of stem cell population maintenance / positive regulation of epithelial cell migration / 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 / actin filament bundle assembly / negative regulation of cell differentiation / brush border / kinesin binding / calyx of Held / actin monomer binding / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / positive regulation of myoblast differentiation / regulation of protein localization to plasma membrane / EPHB-mediated forward signaling / phosphatidylinositol-4,5-bisphosphate binding / substantia nigra development / actin filament polymerization / phosphotyrosine residue binding / axonogenesis / negative regulation of protein binding / neural tube closure / actin filament
Similarity search - Function
Formin, FH3 domain / Formin, GTPase-binding domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Profilin1/2/3, vertebrate / 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 ...Formin, FH3 domain / Formin, GTPase-binding domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Profilin1/2/3, vertebrate / 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. / Profilin conserved 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 / Profilin signature. / Profilin / Profilin / Profilin / Profilin 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 / Armadillo-like helical / Armadillo-type fold / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
: / ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Profilin-1 / Methylated-DNA--protein-cysteine methyltransferase / Actin, cytoplasmic 1 / Cell division control protein 12
Similarity search - Component
Biological speciesHomo sapiens (human)
Schizosaccharomyces pombe (fission yeast)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.25 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 17, 2024Group: Source and taxonomy / Structure summary
Category: em_entity_assembly / em_entity_assembly_molwt ...em_entity_assembly / em_entity_assembly_molwt / em_entity_assembly_naturalsource / em_entity_assembly_recombinant / entity / entity_src_gen / entity_src_nat
Item: _em_entity_assembly.details / _em_entity_assembly.entity_id_list ..._em_entity_assembly.details / _em_entity_assembly.entity_id_list / _em_entity_assembly.name / _em_entity_assembly.source / _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism / _entity.src_method
Revision 1.2Apr 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

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: Methylated-DNA--protein-cysteine methyltransferase,Cell division control protein 12
F: Methylated-DNA--protein-cysteine methyltransferase,Cell division control protein 12
H: Phalloidin (Amanita phalloides)
I: Phalloidin (Amanita phalloides)
J: Phalloidin (Amanita phalloides)
P: Profilin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)341,82521
Polymers339,73410
Non-polymers2,09111
Water0
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 , 3 types, 7 molecules ABCDEFP

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


Mass: 41632.422 Da / Num. of mol.: 4 / Mutation: C272A
Source method: isolated from a genetically manipulated source
Details: Cytoplasmic 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,Cell division control protein 12 / 6-O-methylguanine-DNA methyltransferase / MGMT / O-6-methylguanine-DNA-alkyltransferase


Mass: 77888.820 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Cdc12(FH1FH2) was purified recombinantly from E. coli with a N-terminal snap-tag.
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Schizosaccharomyces pombe (fission yeast)
Gene: MGMT, cdc12, SPAC1F5.04c / Plasmid: pETM-11
Details (production host): The construct contains an N-terminal His10-TEV-SNAP tag
Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Star pRARE
References: UniProt: P16455, UniProt: Q10059, methylated-DNA-[protein]-cysteine S-methyltransferase
#4: Protein Profilin-1 / / Epididymis tissue protein Li 184a / Profilin I


Mass: 15000.207 Da / Num. of mol.: 1 / Mutation: S29C, S71M
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PFN1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Rosetta / References: UniProt: P07737

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Protein/peptide , 1 types, 3 molecules HIJ

#3: Protein/peptide Phalloidin (Amanita phalloides) /


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Phalloidin was bought from sigma. / Source: (natural) Amanita phalloides (death cap) / References: BIRD: PRD_002366

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Non-polymers , 3 types, 11 molecules

#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine 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.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 3 / 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
1Actin-formin-profilin ring complex: the phalloidin-stabilized F-actin barbed end bound by dimeric-Cdc12 and profilin-S71M.COMPLEXHuman beta-actin, human profilin1-S29C/S71M and S. Pombe Cdc12 were purified separately, phalloidin (from Amanita phalloides) was bought from sigma. All components were mixed to assemble the complex prior to cryo-EM grid preparation.#1-#40MULTIPLE SOURCES
2Actin filamentMicrofilamentCOMPLEX#11RECOMBINANT
3Dimeric FH1FH2 domain of S. Pombe Cdc12COMPLEX#21RECOMBINANT
4Profilin-1-S29C/S71MCOMPLEX#41RECOMBINANT
5PhalloidinCOMPLEXStabilizes the actin filament.#31NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Schizosaccharomyces pombe (fission yeast)4896
63Homo sapiens (human)9606
44Homo sapiens (human)9606
55Amanita phalloides (death cap)67723
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellPlasmid
22Trichoplusia ni (cabbage looper)7111BTI-Tnao38
33Escherichia coli (E. coli)562BL21 Star pRAREpETM11
44Escherichia coli (E. coli)562BL21 Rosetta
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 mMsodium chlorideNaClSodium chloride1
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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 3000 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: 63.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5287
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
10cryoSPARCv4.2.1initial 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: 1963686 / Details: Particles picked using SPHIRE-crYOLO.
3D reconstructionResolution: 6.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29092 / 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-Cdc12-phalloidin model1
22PAV

2pav

2PAVprofilin model2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00219668
ELECTRON MICROSCOPYf_angle_d0.55726642
ELECTRON MICROSCOPYf_dihedral_angle_d6.3592678
ELECTRON MICROSCOPYf_chiral_restr0.0412947
ELECTRON MICROSCOPYf_plane_restr0.0033405

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