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- EMDB-19503: Structure of the F-actin barbed end bound by formin mDia1 -

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Entry
Database: EMDB / ID: EMD-19503
TitleStructure of the F-actin barbed end bound by formin mDia1
Map dataSharpened cryo-EM density map of the F-actin barbed end bound by the formin mDia1
Sample
  • Complex: mDia1-bound F-actin barbed end.
    • Complex: Actin filamentMicrofilament
      • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
    • Complex: Mouse mDia1 (FH1FH2C domain)
      • Protein or peptide: Methylated-DNA--protein-cysteine methyltransferase,Protein diaphanous homolog 1
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsactin / formin / Cdc12 / profilin / actin assembly / STRUCTURAL PROTEIN
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
Protein diaphanous homolog 1 / Methylated-DNA--protein-cysteine methyltransferase / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle 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, 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 29, 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_19503.png

Downloads & links

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Map

FileDownload / File: emd_19503.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM density map of the F-actin barbed end bound by the formin mDia1
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.367
Minimum - Maximum-1.222365 - 2.5528903
Average (Standard dev.)0.0025872325 (±0.042644456)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 422.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19503_msk_1.map
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 F-actin...

Fileemd_19503_additional_1.map
Annotation3D-refined, unsharpened cryo-EM density map of the F-actin barbed end bound by the formin mDia1
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 F-actin barbed...

Fileemd_19503_half_map_1.map
AnnotationUnfiltered half map 1 of the F-actin barbed end bound by the formin mDia1
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 F-actin barbed...

Fileemd_19503_half_map_2.map
AnnotationUnfiltered half map 2 of the F-actin barbed end bound by the formin mDia1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : mDia1-bound F-actin barbed end.

EntireName: mDia1-bound F-actin barbed end.
Components
  • Complex: mDia1-bound F-actin barbed end.
    • Complex: Actin filamentMicrofilament
      • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
    • Complex: Mouse mDia1 (FH1FH2C domain)
      • Protein or peptide: Methylated-DNA--protein-cysteine methyltransferase,Protein diaphanous homolog 1
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: mDia1-bound F-actin barbed end.

SupramoleculeName: mDia1-bound F-actin barbed end. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Human beta-actin and mouse mDia1 were purified separately. Both proteins were mixed to assemble the complex prior to cryo-EM grid preparation.

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Supramolecule #2: Actin filament

SupramoleculeName: Actin filament / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Mouse mDia1 (FH1FH2C domain)

SupramoleculeName: Mouse mDia1 (FH1FH2C domain) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)

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

MacromoleculeName: Actin, cytoplasmic 1, N-terminally processed / type: protein_or_peptide / ID: 1
Details: Human beta-actin was recombinantly purified from BTI-Tnao38 cells.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.632422 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: DDDIAALVVD NGSGMCKAGF AGDDAPRAVF PSIVGRPRHQ GVMVGMGQKD SYVGDEAQSK RGILTLKYPI E(HIC)GIVT NWD DMEKIWHHTF YNELRVAPEE HPVLLTEAPL NPKANREKMT QIMFETFNTP AMYVAIQAVL SLYASGRTTG IVMDSGD GV THTVPIYEGY ...String:
DDDIAALVVD NGSGMCKAGF AGDDAPRAVF PSIVGRPRHQ GVMVGMGQKD SYVGDEAQSK RGILTLKYPI E(HIC)GIVT NWD DMEKIWHHTF YNELRVAPEE HPVLLTEAPL NPKANREKMT QIMFETFNTP AMYVAIQAVL SLYASGRTTG IVMDSGD GV THTVPIYEGY ALPHAILRLD LAGRDLTDYL MKILTERGYS FTTTAEREIV RDIKEKLCYV ALDFEQEMAT AASSSSLE K SYELPDGQVI TIGNERFRCP EALFQPSFLG MESAGIHETT FNSIMKCDVD IRKDLYANTV LSGGTTMYPG IADRMQKEI TALAPSTMKI KIIAPPERKY SVWIGGSILA SLSTFQQMWI SKQEYDESGP SIVHRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #2: Methylated-DNA--protein-cysteine methyltransferase,Protein diapha...

MacromoleculeName: Methylated-DNA--protein-cysteine methyltransferase,Protein diaphanous homolog 1
type: protein_or_peptide / ID: 2 / Details: Has a N-terminal snap-tag. / Number of copies: 2 / Enantiomer: LEVO
EC number: methylated-DNA-[protein]-cysteine S-methyltransferase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 86.469258 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASTMDIKLT GEFAMDKDCE MKRTTLDSPL GKLELSGCEQ GLHEIKLLGK GTSAADAVEV PAPAAVLGGP EPLMQATAWL NAYFHQPEA IEEFPVPALH HPVFQQESFT RQVLWKLLKV VKFGEVISYQ QLAALAGNPA ATAAVKTALS GNPVPILIPC H RVVSSSGA ...String:
MASTMDIKLT GEFAMDKDCE MKRTTLDSPL GKLELSGCEQ GLHEIKLLGK GTSAADAVEV PAPAAVLGGP EPLMQATAWL NAYFHQPEA IEEFPVPALH HPVFQQESFT RQVLWKLLKV VKFGEVISYQ QLAALAGNPA ATAAVKTALS GNPVPILIPC H RVVSSSGA VGGYEGGLAV KEWLLAHEGH RLGKPGLGPA GGSPGGGSGG SEMASLSAVV VAPSVSSSAA VPPAPPLPGD SG TVIPPPP PGMGVPPPPP FGFGVPAAPV LPFGLTPKKV YKPEVQLRRP NWSKFVAEDL SQDCFWTKVK EDRFENNELF AKL TLAFSA QTKTSKAKKD QEGGEEKKSV QKKKVKELKV LDSKTAQNLS IFLGSFRMPY QEIKNVILEV NEAVLTESMI QNLI KQMPE PEQLKMLSEL KEEYDDLAES EQFGVVMGTV PRLRPRLNAI LFKLQFSEQV ENIKPEIVSV TAACEELRKS ENFSS LLEL TLLVGNYMNA GSRNAGAFGF NISFLCKLRD TKSADQKMTL LHFLAELCEN DHPEVLKFPD ELAHVEKASR VSAENL QKS LDQMKKQIAD VERDVQNFPA ATDEKDKFVE KMTSFVKDAQ EQYNKLRMMH SNMETLYKEL GDYFVFDPKK LSVEEFF MD LHNFRNMFLQ AVKENQKRRE TEEKMRRAKL AKEKAEKERL EKQQKREQLI DMNAEGDETG VMDSLLEALQ SGAAFRRK R GPRQVNRKAG CAVTSLLASE LTKDDAMAPG PVKVPKKSEG VPTILEEAKE LVGRASHHHH HH

UniProtKB: Methylated-DNA--protein-cysteine methyltransferase, Protein diaphanous homolog 1, Protein diaphanous homolog 1

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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: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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
10.0 mMHEPES
70.0 mMpotassium chlorideKCl
0.01 % (v/v)Tween20
0.5 mMTCEP
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: 0.01 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
Specialist opticsSpherical aberration corrector: Titan Krios G2 microscope (Thermo Fisher Scientific) with an in-column Cs-corrector.
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 G2 microscope (Thermo Fisher Scientific) with an in-column Cs-corrector.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 38913 / Average electron dose: 67.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1963686 / Details: Particles picked using SPHIRE-crYOLO.
Startup modelType of model: OTHER / Details: Ab initio reconstruction in CryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.3.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v4.3.0) / Details: 2D classification in cryoSPARC.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.3.0) / Number images used: 150807
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

8rtt

source_name: PDB, initial_model_type: experimental modelActin model

3obv

source_name: PDB, initial_model_type: experimental modelmDia1 model
DetailsRefinement performed using phenix real-space refine
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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