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- PDB-8rtt: Structure of the formin Cdc12 bound to the barbed end of phalloid... -

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Basic information

Entry
Database: PDB / ID: 8rtt
TitleStructure of the formin Cdc12 bound to the barbed end of phalloidin-stabilized F-actin.
Components
  • Actin, cytoplasmic 1, N-terminally processed
  • Cell division control protein 12
  • Phalloidin
KeywordsSTRUCTURAL PROTEIN / actin / formin / Cdc12 / actin assembly.
Function / homology
Function and homology information


protein localization to mitotic actomyosin contractile ring / F-bar domain binding / medial cortical node / mitotic actomyosin contractile ring, proximal layer / mitotic actomyosin contractile ring / medial cortex / mitotic actomyosin contractile ring assembly / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex ...protein localization to mitotic actomyosin contractile ring / F-bar domain binding / medial cortical node / mitotic actomyosin contractile ring, proximal layer / mitotic actomyosin contractile ring / medial cortex / mitotic actomyosin contractile ring assembly / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / nBAF complex / brahma complex / regulation of transepithelial transport / Formation of annular gap junctions / morphogenesis of a polarized epithelium / Formation of the dystrophin-glycoprotein complex (DGC) / structural constituent of postsynaptic actin cytoskeleton / Gap junction degradation / barbed-end actin filament capping / GBAF complex / Folding of actin by CCT/TriC / regulation of G0 to G1 transition / protein localization to adherens junction / Cell-extracellular matrix interactions / dense body / postsynaptic actin cytoskeleton / Tat protein binding / mating projection tip / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / regulation of double-strand break repair / regulation of nucleotide-excision repair / Adherens junctions interactions / RHOF GTPase cycle / adherens junction assembly / apical protein localization / Sensory processing of sound by outer hair cells of the cochlea / Interaction between L1 and Ankyrins / tight junction / 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 / apical junction complex / cell division site / positive regulation of double-strand break repair / regulation of norepinephrine uptake / transporter regulator activity / maintenance of blood-brain barrier / nitric-oxide synthase binding / cortical cytoskeleton / NuA4 histone acetyltransferase complex / establishment or maintenance of cell polarity / positive regulation of stem cell population maintenance / Regulation of MITF-M-dependent genes involved in pigmentation / Recycling pathway of L1 / brush border / actin filament bundle assembly / regulation of G1/S transition of mitotic cell cycle / EPH-ephrin mediated repulsion of cells / kinesin binding / negative regulation of cell differentiation / RHO GTPases Activate WASPs and WAVEs / regulation of synaptic vesicle endocytosis / positive regulation of myoblast differentiation / RHO GTPases activate IQGAPs / regulation of protein localization to plasma membrane / positive regulation of double-strand break repair via homologous recombination / actin filament polymerization / EPHB-mediated forward signaling / cytoskeleton organization / substantia nigra development / axonogenesis / calyx of Held / nitric-oxide synthase regulator activity / Translocation of SLC2A4 (GLUT4) to the plasma membrane / FCGR3A-mediated phagocytosis / actin filament / adherens junction / positive regulation of cell differentiation / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / cell motility / RHO GTPases Activate Formins / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / DNA Damage Recognition in GG-NER / kinetochore / Regulation of actin dynamics for phagocytic cup formation / B-WICH complex positively regulates rRNA expression / structural constituent of cytoskeleton / small GTPase binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / platelet aggregation / VEGFA-VEGFR2 Pathway / tau protein binding / Schaffer collateral - CA1 synapse / nuclear matrix / cytoplasmic ribonucleoprotein granule / Signaling by RAF1 mutants
Similarity search - Function
: / 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. / Formin, FH2 domain ...: / 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. / Formin, FH2 domain / Formin, FH2 domain superfamily / Formin Homology 2 Domain / Formin homology-2 (FH2) domain profile. / Formin Homology 2 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
Similarity search - Domain/homology
: / ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / 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: 3.56 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 1.1Jun 5, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _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: Cell division control protein 12
H: Phalloidin
I: Phalloidin
J: Phalloidin
F: Cell division control protein 12
hetero molecules


Theoretical massNumber of molelcules
Total (without water)268,56320
Polymers266,4729
Non-polymers2,09111
Water00
1


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

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Components

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Protein , 2 types, 6 molecules ABCDEF

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


Mass: 41763.617 Da / Num. of mol.: 4 / Mutation: C272A
Source method: isolated from a genetically manipulated source
Details: Recombinant beta-actin 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 Cell division control protein 12


Mass: 48495.242 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: FH2 domain of S. pombe Cdc12, purified from E. coli cells.
Source: (gene. exp.) Schizosaccharomyces pombe (fission yeast)
Gene: cdc12, SPAC1F5.04c / Plasmid: pETM11-SUMO3
Details (production host): Contains N-terminal His10-Sumo tag.
Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Star pRARE / References: UniProt: Q10059

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Protein/peptide , 1 types, 3 molecules HIJ

#3: Protein/peptide Phalloidin


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: phalloidin, purified from Amanita phalloides. / Source: (natural) Amanita phalloides (death cap) / Plasmid details: Bought from sigma. / References: BIRD: PRD_002366

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Non-polymers , 3 types, 11 molecules

#4: 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
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4

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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
1Complex of the dimeric FH2 domain of S. Pombe Cdc12 bound to the barbed end of phalloidin stabilized F-actin.COMPLEXHuman beta-actin 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-#30MULTIPLE SOURCES
2Actin filamentCOMPLEX#11RECOMBINANT
3Dimeric FH2 domain of S. Pombe Cdc12COMPLEX#21RECOMBINANT
4Phalloidin.COMPLEXStabilizes the actin filament.#31NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Schizosaccharomyces pombe (fission yeast)4896
44Amanita phalloides (death cap)67723
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellPlasmid
22Trichoplusia ni (cabbage looper)7111BTI-Tnao38
33Escherichia coli (E. coli)562BL21 Star pRAREpETM11-SUMO3
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 chlorideNaCl1
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
11RELION3.1.0final Euler assignment
12RELION3.1.0classification
13RELION3.1.03D 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: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154570 / Details: Refinement performed in RELION. / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDDetailsInitial refinement model-IDSource nameType
18A2S

8a2s

C8A2SCactin model1PDBexperimental model
2Cdc12 modelAlphaFoldin silico model
36T1Y

6t1y

6T1Yphalloidin model3PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 63.41 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002118539
ELECTRON MICROSCOPYf_angle_d0.471425110
ELECTRON MICROSCOPYf_chiral_restr0.0392774
ELECTRON MICROSCOPYf_plane_restr0.00293208
ELECTRON MICROSCOPYf_dihedral_angle_d5.73422519

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