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- EMDB-19522: Structure of the formin INF2 bound to the barbed end of F-actin. -

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Entry
Database: EMDB / ID: EMD-19522
TitleStructure of the formin INF2 bound to the barbed end of F-actin.
Map dataSharpened, local-resolution filtered cryo-EM density map of the formin INF2 bound to the barbed end of actin filaments.
Sample
  • Complex: Complex of the formin INF2 dimer that binds to the actin subunits at the barbed end of actin filaments.
    • Complex: Actin filament barbed end
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: INF2 dimer
      • Protein or peptide: Isoform 2 of Inverted formin-2
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Keywordsactin / formin / INF2 / actin end / barbed end / STRUCTURAL PROTEIN
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
Actin, alpha skeletal muscle / Inverted formin-2
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Homo sapiens (human)
Methodsingle 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, 3 items
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, 2024-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19522.png

Downloads & links

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Map

FileDownload / File: emd_19522.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened, local-resolution filtered cryo-EM density map of the formin INF2 bound to the barbed end of actin filaments.
Voxel sizeX=Y=Z: 0.9 Å
Density
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.12075663 - 0.21737441
Average (Standard dev.)0.00013351534 (±0.003831739)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19522_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_19522_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: 3D-refined, unsharpened cryo-EM density map of INF2 bound...

Fileemd_19522_additional_1.map
Annotation3D-refined, unsharpened cryo-EM density map of INF2 bound to the barbed end of F-actin. This reconstruction was computed with a subset of particles that displayed better density for the FH2L.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered half map 1 of the formin INF2...

Fileemd_19522_additional_2.map
AnnotationUnfiltered half map 1 of the formin INF2 bound to the barbed end of actin filaments. This reconstruction was computed with a subset of particles that displayed better density for the FH2L.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered half map 2 of the formin INF2...

Fileemd_19522_additional_3.map
AnnotationUnfiltered half map 2 of the formin INF2 bound to the barbed end of actin filaments. This reconstruction was computed with a subset of particles that displayed better density for the FH2L.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D-refined, unsharpened cryo-EM density map of the formin...

Fileemd_19522_additional_4.map
Annotation3D-refined, unsharpened cryo-EM density map of the formin INF2 bound to the barbed end of actin filaments.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened cryo-EM density map of the formin INF2...

Fileemd_19522_additional_5.map
AnnotationSharpened cryo-EM density map of the formin INF2 bound to the barbed end of actin filaments. This reconstruction was computed with a subset of particles that displayed better density for the FH2L.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Composite map computed with the main map of...

Fileemd_19522_additional_6.map
AnnotationComposite map computed with the main map of INF2 bound to the barbed end of F-actin and a secondary map where the FH2L is better resolved. This map is intended for visualization purposes.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 1 of the formin INF2...

Fileemd_19522_half_map_1.map
AnnotationUnfiltered half map 1 of the formin INF2 bound to the barbed end of actin filaments.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 2 of the formin INF2...

Fileemd_19522_half_map_2.map
AnnotationUnfiltered half map 2 of the formin INF2 bound to the barbed end of actin filaments.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of the formin INF2 dimer that binds to the actin subunits...

EntireName: Complex of the formin INF2 dimer that binds to the actin subunits at the barbed end of actin filaments.
Components
  • Complex: Complex of the formin INF2 dimer that binds to the actin subunits at the barbed end of actin filaments.
    • Complex: Actin filament barbed end
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: INF2 dimer
      • Protein or peptide: Isoform 2 of Inverted formin-2
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Complex of the formin INF2 dimer that binds to the actin subunits...

SupramoleculeName: Complex of the formin INF2 dimer that binds to the actin subunits at the barbed end of actin filaments.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Alpha 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.

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Supramolecule #2: Actin filament barbed end

SupramoleculeName: Actin filament barbed end / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Complex of the actin subunits that form the barbed end of actin filaments.
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Skeletal muscle

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Supramolecule #3: INF2 dimer

SupramoleculeName: INF2 dimer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: This dimer is composed of the FH2 domain of the formin INF2.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1
Details: Rabbit skeletal alpha actin purified from frozen rabbit muscle acetone powder.
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Skeletal muscle
Molecular weightTheoretical: 41.875633 KDa
SequenceString: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG ...String:
DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: Isoform 2 of Inverted formin-2

MacromoleculeName: Isoform 2 of Inverted formin-2 / type: protein_or_peptide / ID: 2
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.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 84.453711 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GGGGGMAPPA PPLPPPLPGS CEFLSPPPPP LPGLGCPPPP PPLLPGMGWG PPPPPPPLLP CTCSPPVAGG MEEVIVAQVD HGLGSAWVP SHRRVNPPTL RMKKLNWQKL PSNVAREHNS MWASLSSPDA EAVEPDFSSI ERLFSFPAAK PKEPTMVAPR A RKEPKEIT ...String:
GGGGGMAPPA PPLPPPLPGS CEFLSPPPPP LPGLGCPPPP PPLLPGMGWG PPPPPPPLLP CTCSPPVAGG MEEVIVAQVD HGLGSAWVP SHRRVNPPTL RMKKLNWQKL PSNVAREHNS MWASLSSPDA EAVEPDFSSI ERLFSFPAAK PKEPTMVAPR A RKEPKEIT FLDAKKSLNL NIFLKQFKCS NEEVAAMIRA GDTTKFDVEV LKQLLKLLPE KHEIENLRAF TEERAKLASA DH FYLLLLA IPCYQLRIEC MLLCEGAAAV LDMVRPKAQL VLAACESLLT SRQLPIFCQL ILRIGNFLNY GSHTGDADGF KIS TLLKLT ETKSQQNRVT LLHHVLEEAE KSHPDLLQLP RDLEQPSQAA GINLEIIRSE ASSNLKKLLE TERKVSASVA EVQE QYTER LQASISAFRA LDELFEAIEQ KQRELADYLC EDAQQLSLED TFSTMKAFRD LFLRALKENK DRKEQAAKAE RRKQQ LAEE EARRPRGEDG KPVRKGPGKQ EEVCVIDALL ADIRKGFQLR KTARGRGDTD GGSKAASMDP PRATEPVATS NPAGDP VGS TRCPASEPGL DATTASESRG WDLVDAVTPG PQPTLEQLEE GGPRPLERRS SWYVDASDVL TTEDPQCPQP LEGAWPV TL GDAQALKPLK FSSNQPPAAG SSRQDAKDPT SLLGVLQAEA DSTSEGLEDA VHSRGARPPA AGPGGDEDED EEDTAPES A LDTSLDKSFS EDAVTDSSGS GTLPRARGRA SKGTGKRRKK RPSRSQEGLR PRPKAK

UniProtKB: Inverted formin-2

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Macromolecule #6: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.1
Component:
ConcentrationNameFormula
12.0 mMHEPES
100.0 mMpotassium chlorideKCl
2.1 mMmagnesium chlorideMgCl2
1.0 mMEGTA
1.0 mMTCEP
0.2 mMATPAdenosine triphosphate

Details: 12 mM HEPES pH 7.1, 100 mM KCl, 2.1 mM MgCl2, 1 mM EGTA, 1 mM TCEP, 0.2 mM ATP
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV / Details: 3 seconds, force 0..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 105000
Specialist opticsSpherical aberration corrector: 300 kV Titan Krios G3 microscope (Thermo Fisher Scientific).
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV / Details: Gatan energy filter.
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Details300 kV Titan Krios G3 microscope (Thermo Fisher Scientific).
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 20305 / Average electron dose: 60.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1935707 / Details: Particles picked using SPHIRE-crYOLO.
Startup modelType of model: EMDB MAP
EMDB ID:

3835

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.1.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v4.1.1) / Details: 2D classification in cryoSPARC.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 142549
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

8a2s

source_name: PDB, initial_model_type: experimental modelactin model
source_name: AlphaFold, initial_model_type: in silico modelINF2 model
DetailsReal Space Refinement in Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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