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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 filament
      • 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 / ERBB2 Regulates Cell Motility / RHOF GTPase cycle / MGMT-mediated DNA damage reversal / RHOC GTPase cycle / RHOD GTPase cycle / actin nucleation / neuron projection retraction / methylated-DNA-[protein]-cysteine S-methyltransferase ...negative regulation of neuron projection regeneration / multicellular organismal locomotion / ERBB2 Regulates Cell Motility / RHOF GTPase cycle / MGMT-mediated DNA damage reversal / RHOC GTPase cycle / RHOD GTPase cycle / actin nucleation / neuron projection retraction / methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / RHOB GTPase cycle / RHO GTPases Activate Formins / positive regulation of norepinephrine uptake / RHOA GTPase cycle / DNA-methyltransferase activity / profilin binding / 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 / 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 / regulation of microtubule-based process / DNA alkylation repair / 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 / axon midline choice point recognition / 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 / 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 / regulation of G1/S transition of mitotic cell cycle / EPH-ephrin mediated repulsion of cells / kinesin binding / negative regulation of cell differentiation / synaptic vesicle endocytosis / RHO GTPases Activate WASPs and WAVEs / regulation of synaptic vesicle endocytosis / ephrin receptor signaling pathway / 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 / Neutrophil degranulation / EPHB-mediated forward signaling / cytoskeleton organization / substantia nigra development / axonogenesis / calyx of Held / nitric-oxide synthase regulator activity / methyltransferase 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 / sensory perception of sound / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / DNA Damage Recognition in GG-NER
Similarity search - Function
DRF autoregulatory / DRF Autoregulatory 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 ...DRF autoregulatory / DRF Autoregulatory 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. / Formin, FH2 domain / Formin, FH2 domain superfamily / Formin Homology 2 Domain / Formin homology-2 (FH2) domain profile. / Formin Homology 2 Domain / Methylguanine DNA methyltransferase, ribonuclease-like domain / 6-O-methylguanine DNA methyltransferase, ribonuclease-like 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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 480 pix.
= 422.4 Å		0.88 Å/pix.
x 480 pix.
= 422.4 Å		0.88 Å/pix.
x 480 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88 Å
Density

Histogram

Histogram (log scale)
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 filament
      • 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

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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
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.
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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
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
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.3.0)
Final 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.
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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