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- PDB-8rv2: Structure of the formin INF2 bound to the barbed end of F-actin. -

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Basic information

Entry
Database: PDB / ID: 8rv2
TitleStructure of the formin INF2 bound to the barbed end of F-actin.
Components
  • Actin, alpha skeletal muscle
  • Isoform 2 of Inverted formin-2
KeywordsSTRUCTURAL PROTEIN / actin / formin / INF2 / actin end / barbed end
Function / homology
Function and homology information


cytoskeletal motor activator activity / regulation of mitochondrial fission / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly ...cytoskeletal motor activator activity / regulation of mitochondrial fission / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / small GTPase binding / calcium-dependent protein binding / lamellipodium / cell body / actin binding / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / perinuclear region of cytoplasm / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Inverted formin-2 / 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 ...Inverted formin-2 / 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 / WH2 motif / WH2 domain / WH2 domain profile. / 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 / ADENOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / Actin, alpha skeletal muscle / Inverted formin-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.41 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 31, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Database references / Structure summary / Category: citation / citation_author / em_entity_assembly
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 / _em_entity_assembly.source

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
E: Isoform 2 of Inverted formin-2
F: Isoform 2 of Inverted formin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)338,39115
Polymers336,4106
Non-polymers1,9819
Water0
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, alpha skeletal muscle / / Alpha-actin-1


Mass: 41875.633 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Details: Rabbit skeletal alpha actin purified from frozen rabbit muscle acetone powder.
Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: Skeletal muscle / References: UniProt: P68135
#2: Protein Isoform 2 of Inverted formin-2 / HBEBP2-binding protein C


Mass: 84453.711 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: INF2 FH1FH2C nonCAAX isoform was recombinantly expressed in E. coli BL21 Star pRARE cells and purified. This compound consists of the formin homology 1 and 2 domains, as well as the C- ...Details: INF2 FH1FH2C nonCAAX isoform was recombinantly expressed in E. coli BL21 Star pRARE cells and purified. This compound consists of the formin homology 1 and 2 domains, as well as the C-terminal region of the human formin INF2 isoform nonCAAX.
Source: (gene. exp.) Homo sapiens (human) / Gene: INF2, C14orf151, C14orf173 / Plasmid: pETMz2 expression vector
Details (production host): Insert: His6-Z-TEV-Gly5-INF2(FH1FH2C)
Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Star pRARE / References: UniProt: Q27J81

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Non-polymers , 4 types, 9 molecules

#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.: 4 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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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 formin INF2 dimer that binds to the actin subunits at the barbed end of actin filaments.COMPLEXAlpha actin was purified from rabbit skeletal muscle. INF2 was purified from E. coli. Actin filaments were co-polymerized in the presence of the formin INF2 prior to cryo-EM grid preparation.#1-#20MULTIPLE SOURCES
2Actin filament barbed endCOMPLEXComplex of the actin subunits that form the barbed end of actin filaments.#11NATURAL
3INF2 dimerCOMPLEXThis dimer is composed of the FH2 domain of the formin INF2.#21RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
12NO
23NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDTissue
12Oryctolagus cuniculus (rabbit)9986Skeletal muscle
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Trichoplusia ni (cabbage looper)7111
23Escherichia coli (E. coli)562BL21 Star pRARE
Buffer solutionpH: 7.1
Details: 12 mM HEPES pH 7.1, 100 mM KCl, 2.1 mM MgCl2, 1 mM EGTA, 1 mM TCEP, 0.2 mM ATP
Buffer component
IDConc.NameFormulaBuffer-ID
112 mMHEPES1
2100 mMpotassium chlorideKCl1
32.1 mMmagnesium chlorideMgCl21
41 mMEGTA1
51 mMTCEP1
60.2 mMATPAdenosine triphosphate1
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 G3 microscope (Thermo Fisher Scientific).
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2900 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 20305
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: Gatan energy filter. / Energyfilter slit width: 15 eV
Spherical aberration corrector: 300 kV Titan Krios G3 microscope (Thermo Fisher Scientific).

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Processing

EM software
IDNameVersionCategory
1crYOLO1.8particle selection
2EPUimage acquisition
4CTFFIND4.13CTF correction
7UCSF ChimeraX1.5model fitting
8Coot0.9.8.1model fitting
10cryoSPARCv4.1.0initial Euler assignment
11RELION3.1.0final Euler assignment
12cryoSPARCv4.1.1classification
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: 1935707 / Details: Particles picked using SPHIRE-crYOLO.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142549 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: Real Space Refinement in Phenix.
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeDetailsInitial refinement model-IDSource nameType
18A2S

8a2s

8A2Sactin model1PDBexperimental model
2INF2 modelAlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00217585
ELECTRON MICROSCOPYf_angle_d0.45423811
ELECTRON MICROSCOPYf_dihedral_angle_d6.5512395
ELECTRON MICROSCOPYf_chiral_restr0.0392680
ELECTRON MICROSCOPYf_plane_restr0.0033047

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