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- EMDB-44135: Dia1 in the Middle of F-actin -

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Basic information

Entry
Database: EMDB / ID: EMD-44135
TitleDia1 in the Middle of F-actin
Map data
Sample
  • Complex: Structure of mDia1 dimer bound at the barbed end of actin filaments.
    • Complex: mDia1 dimer
      • Protein or peptide: Protein diaphanous homolog 1
    • Complex: Actin filamentMicrofilament
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsActin / Filament / Elongation / Ends / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


negative regulation of neuron projection regeneration / multicellular organismal locomotion / RHOF GTPase cycle / RHOB GTPase cycle / ERBB2 Regulates Cell Motility / RHOC GTPase cycle / RHOD GTPase cycle / RHOA GTPase cycle / actin nucleation / neuron projection retraction ...negative regulation of neuron projection regeneration / multicellular organismal locomotion / RHOF GTPase cycle / RHOB GTPase cycle / ERBB2 Regulates Cell Motility / RHOC GTPase cycle / RHOD GTPase cycle / RHOA GTPase cycle / actin nucleation / neuron projection retraction / RHO GTPases Activate Formins / profilin binding / regulation of microtubule-based process / axon midline choice point recognition / cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / myosin heavy chain binding / troponin I binding / filamentous actin / actin filament bundle / brush border / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / synaptic vesicle endocytosis / skeletal muscle myofibril / actin monomer binding / ephrin receptor signaling pathway / skeletal muscle fiber development / stress fiber / titin binding / cytoskeleton organization / actin filament polymerization / Neutrophil degranulation / filopodium / actin filament / sensory perception of sound / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / protein localization / brain development / small GTPase binding / ruffle membrane / spindle / calcium-dependent protein binding / neuron projection development / presynapse / lamellipodium / actin binding / gene expression / regulation of cell shape / cell body / actin cytoskeleton organization / transmembrane transporter binding / hydrolase activity / positive regulation of cell migration / neuron projection / protein domain specific binding / centrosome / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / nucleus / cytoplasm
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. / 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
Protein diaphanous homolog 1 / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.41 Å
AuthorsPalmer NJ / Barrie KR / Dominguez R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nature / Year: 2024
Title: Mechanisms of actin filament severing and elongation by formins.
Authors: Nicholas J Palmer / Kyle R Barrie / Roberto Dominguez /
Abstract: Humans express fifteen formins, playing crucial roles in actin-based processes, such as cytokinesis, cell motility, and mechanotransduction . However, the lack of structures bound to the actin ...Humans express fifteen formins, playing crucial roles in actin-based processes, such as cytokinesis, cell motility, and mechanotransduction . However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. While formins are known for their ability to nucleate and elongate F-actin , some formins can additionally depolymerize, sever, or bundle F-actin. Two mammalian formins, inverted formin-2 (INF2) and diaphanous-1 (Dia1), exemplify this diversity. INF2 displays potent severing activity but elongates weakly , whereas Dia1 has potent elongation activity but does not sever . Using cryo-electron microscopy (cryo-EM), we reveal five structural states of INF2 and two of Dia1 bound to the middle and barbed end of F-actin. INF2 and Dia1 bind differently to these sites, consistent with their distinct activities. The FH2 and WH2 domains of INF2 are positioned to sever F-actin, whereas Dia1 appears unsuited for severing. Structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.
History
DepositionMar 19, 2024-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44135.png

Downloads & links

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Map

FileDownload / File: emd_44135.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.126
Minimum - Maximum-0.23665002 - 0.62124634
Average (Standard dev.)0.0024956109 (±0.02510415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44135_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44135_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44135_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of mDia1 dimer bound at the barbed end of actin filaments.

EntireName: Structure of mDia1 dimer bound at the barbed end of actin filaments.
Components
  • Complex: Structure of mDia1 dimer bound at the barbed end of actin filaments.
    • Complex: mDia1 dimer
      • Protein or peptide: Protein diaphanous homolog 1
    • Complex: Actin filamentMicrofilament
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Structure of mDia1 dimer bound at the barbed end of actin filaments.

SupramoleculeName: Structure of mDia1 dimer bound at the barbed end of actin filaments.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 252 KDa

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Supramolecule #2: mDia1 dimer

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

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Supramolecule #3: Actin filament

SupramoleculeName: Actin filament / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 41.387227 KDa
SequenceString: TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIE(HIC)G IITNWD DME KIWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVT HN VPIYEGYALP ...String:
TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIE(HIC)G IITNWD DME KIWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVT HN VPIYEGYALP HAIMRLDLAG RDLTDYLMKI LTERGYSFVT TAEREIVRDI KEKLCYVALD FENEMATAAS SSSLEKSY E LPDGQVITIG NERFRCPETL FQPSFIGMES AGIHETTYNS IMKCDIDIRK DLYANNVMSG GTTMYPGIAD RMQKEITAL APSTMKIKII APPERKYSVW IGGSILASLS TFQQMWITKQ EYDEAGPSIV HRKCF

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: Protein diaphanous homolog 1

MacromoleculeName: Protein diaphanous homolog 1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 46.313004 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: VLPFGLTPKK VYKPEVQLRR PNWSKFVAED LSQDCFWTKV KEDRFENNEL FAKLTLAFSA QTKTSKAKKD QEGGEEKKSV QKKKVKELK VLDSKTAQNL SIFLGSFRMP YQEIKNVILE VNEAVLTESM IQNLIKQMPE PEQLKMLSEL KEEYDDLAES E QFGVVMGT ...String:
VLPFGLTPKK VYKPEVQLRR PNWSKFVAED LSQDCFWTKV KEDRFENNEL FAKLTLAFSA QTKTSKAKKD QEGGEEKKSV QKKKVKELK VLDSKTAQNL SIFLGSFRMP YQEIKNVILE VNEAVLTESM IQNLIKQMPE PEQLKMLSEL KEEYDDLAES E QFGVVMGT VPRLRPRLNA ILFKLQFSEQ VENIKPEIVS VTAACEELRK SENFSSLLEL TLLVGNYMNA GSRNAGAFGF NI SFLCKLR DTKSADQKMT LLHFLAELCE NDHPEVLKFP DELAHVEKAS RVSAENLQKS LDQMKKQIAD VERDVQNFPA ATD EKDKFV EKMTSFVKDA QEQYNKLRMM HSNMETLYKE LGDYFVFDPK KLSVEEFFMD LHNFRNMFLQ AVKENQKRRE TEE

UniProtKB: Protein diaphanous homolog 1

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / 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: 6 / 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

Concentration1.48 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMHEPES
50.0 mMNaClSodium chloride
1.0 mMEDTAEthylenediaminetetraacetic acid
1.0 mMDTT
0.05 %Thesit

Details: 20mM HEPES, 50mM NaCL, 1mM EDTA, 1mM DTT, 0.05% Thesit
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 100 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsActin filaments were sonicated prior to addition of mDia1

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8f8p


Details: Actin filament from 8F8P, mDia1 from alphafold model
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0)
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 59861
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

8f8p

source_name: PDB, initial_model_type: experimental model
source_name: AlphaFold, initial_model_type: in silico modelMultimer
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9b3d:
mDia1 in the middle of F-actin

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