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- EMDB-19497: Cryo-EM reconstruction of the formin Cdc12 bound to the barbed en... -

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Entry
Database: EMDB / ID: EMD-19497
TitleCryo-EM reconstruction of the formin Cdc12 bound to the barbed end of F-actin (without phalloidin)
Map dataSharpened cryo-EM density map of the formin Cdc12 bound to the barbed end of F-actin (without phalloidin).
Sample
  • Complex: Complex of the dimeric FH2 domain of S. Pombe Cdc12 bound to the barbed end of F-actin.
    • Complex: Actin filament
      • Protein or peptide: human cytoplasmic beta-actin
    • Complex: Dimeric FH2 domain of S. Pombe Cdc12
      • Protein or peptide: S. pombe Cdc12
Keywordsactin / formin / Cdc12 / actin assembly. / STRUCTURAL PROTEIN
Function / homology
Function and homology information


protein localization to mitotic actomyosin contractile ring / F-bar domain binding / medial cortical node / mitotic actomyosin contractile ring, proximal layer / mitotic actomyosin contractile ring / medial cortex / mitotic actomyosin contractile ring assembly / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / Formation of the embryonic stem cell BAF (esBAF) complex ...protein localization to mitotic actomyosin contractile ring / F-bar domain binding / medial cortical node / mitotic actomyosin contractile ring, proximal layer / mitotic actomyosin contractile ring / medial cortex / mitotic actomyosin contractile ring assembly / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / Formation of the embryonic stem cell BAF (esBAF) complex / regulation of transepithelial transport / Formation of the canonical BAF (cBAF) complex / mating projection tip / morphogenesis of a polarized epithelium / Formation of annular gap junctions / Formation of the dystrophin-glycoprotein complex (DGC) / Formation of the polybromo-BAF (pBAF) complex / structural constituent of postsynaptic actin cytoskeleton / Gap junction degradation / GBP-mediated host defense / Formation of neuronal progenitor and neuronal BAF (npBAF and nBAF) / protein localization to adherens junction / Formation of the non-canonical BAF (ncBAF) complex / barbed-end actin filament capping / Cell-extracellular matrix interactions / regulation of G0 to G1 transition / dense body / Tat protein binding / Folding of actin by CCT/TriC / postsynaptic actin cytoskeleton / Regulation of CDH1 Function / apical protein localization / Adherens junctions interactions / microtubule nucleation / Prefoldin mediated transfer of substrate to CCT/TriC / RHOF GTPase cycle / adherens junction assembly / regulation of double-strand break repair / Sensory processing of sound by outer hair cells of the cochlea / tight junction / Sensory processing of sound by inner hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / positive regulation of T cell differentiation / maintenance of blood-brain barrier / Interaction between L1 and Ankyrins / apical junction complex / regulation of nucleotide-excision repair / positive regulation of stem cell population maintenance / NuA4 histone acetyltransferase complex / regulation of norepinephrine uptake / cell division site / transporter regulator activity / Recycling pathway of L1 / positive regulation of double-strand break repair / cortical cytoskeleton / Regulation of MITF-M-dependent genes involved in pigmentation / negative regulation of cell differentiation / establishment or maintenance of cell polarity / nitric-oxide synthase binding / brush border / actin filament bundle assembly / EPH-ephrin mediated repulsion of cells / regulation of synaptic vesicle endocytosis / RHO GTPases Activate WASPs and WAVEs / positive regulation of myoblast differentiation / kinesin binding / regulation of protein localization to plasma membrane / RHO GTPases activate IQGAPs / positive regulation of double-strand break repair via homologous recombination / regulation of G1/S transition of mitotic cell cycle / axonogenesis / cytoskeleton organization / actin filament polymerization / EPHB-mediated forward signaling / substantia nigra development / calyx of Held / cell motility / nitric-oxide synthase regulator activity / FCGR3A-mediated phagocytosis / actin filament / Translocation of SLC2A4 (GLUT4) to the plasma membrane / adherens junction / positive regulation of cell differentiation / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / RHO GTPases Activate Formins / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / Regulation of actin dynamics for phagocytic cup formation / platelet aggregation / VEGFA-VEGFR2 Pathway / B-WICH complex positively regulates rRNA expression / tau protein binding / Schaffer collateral - CA1 synapse / DNA Damage Recognition in GG-NER / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / small GTPase binding / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / kinetochore / nuclear matrix
Similarity search - Function
: / 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, 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
Actin, cytoplasmic 1 / Cell division control protein 12
Similarity search - Component
Biological speciesHomo sapiens (human) / Schizosaccharomyces pombe (fission yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.54 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_19497.png

Downloads & links

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Map

FileDownload / File: emd_19497.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM density map of the formin Cdc12 bound to the barbed end of F-actin (without phalloidin).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 320 pix.
= 281.6 Å		0.88 Å/pix.
x 320 pix.
= 281.6 Å		0.88 Å/pix.
x 320 pix.
= 281.6 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: 0.01
Minimum - Maximum-0.00765671 - 0.03492895
Average (Standard dev.)0.00026118013 (±0.0015996065)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19497_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 formin...

Fileemd_19497_additional_1.map
Annotation3D-refined, unsharpened cryo-EM density map of the formin Cdc12 bound to the barbed end of F-actin (without phalloidin).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-EM map Cdc12 bound to phalloidin-stabilized F-actin (EMD-19496),...

Fileemd_19497_additional_2.map
AnnotationCryo-EM map Cdc12 bound to phalloidin-stabilized F-actin (EMD-19496), resampled on the cryo-EM reconstruction that was obtained without phalloidin.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Power-adjusted map of actin-Cdc12 with phalloidin(EMD-19496), resampled on...

Fileemd_19497_additional_3.map
AnnotationPower-adjusted map of actin-Cdc12 with phalloidin(EMD-19496), resampled on the no-phalloidin map. Used to construct a difference map between the reconstructions with and without phalloidin.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: Difference map between actin-Cdc12 reconstructions that were obtained...

Fileemd_19497_additional_4.map
AnnotationDifference map between actin-Cdc12 reconstructions that were obtained in the absence (this entry) and presence (EMD-19496) of the toxin phalloidin.
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 Cdc12...

Fileemd_19497_half_map_1.map
AnnotationUnfiltered half map 1 of the formin Cdc12 bound to the barbed end of F-actin (without phalloidin).
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 Cdc12...

Fileemd_19497_half_map_2.map
AnnotationUnfiltered half map 2 of the formin Cdc12 bound to the barbed end of F-actin (without phalloidin).
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 dimeric FH2 domain of S. Pombe Cdc12 bound to the ...

EntireName: Complex of the dimeric FH2 domain of S. Pombe Cdc12 bound to the barbed end of F-actin.
Components
  • Complex: Complex of the dimeric FH2 domain of S. Pombe Cdc12 bound to the barbed end of F-actin.
    • Complex: Actin filament
      • Protein or peptide: human cytoplasmic beta-actin
    • Complex: Dimeric FH2 domain of S. Pombe Cdc12
      • Protein or peptide: S. pombe Cdc12

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Supramolecule #1: Complex of the dimeric FH2 domain of S. Pombe Cdc12 bound to the ...

SupramoleculeName: Complex of the dimeric FH2 domain of S. Pombe Cdc12 bound to the barbed end of F-actin.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Human beta-actin and S. Pombe Cdc12 were purified separately. Both proteins were mixed to assemble the complex prior to cryo-EM grid preparation.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Actin filament

SupramoleculeName: Actin filament / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: Dimeric FH2 domain of S. Pombe Cdc12

SupramoleculeName: Dimeric FH2 domain of S. Pombe Cdc12 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)

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Macromolecule #1: human cytoplasmic beta-actin

MacromoleculeName: human cytoplasmic beta-actin / type: protein_or_peptide / ID: 1
Details: Actin purified recombinantly from BTI-Tnao38 cells.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIVTNWD DMEKIWHHTF YNELRVAPEE HPVLLTEAPL NPKANREKMT QIMFETFNTP AMYVAIQAVL SLYASGRTTG IVMDSGDGVT HTVPIYEGYA ...String:
MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIVTNWD DMEKIWHHTF YNELRVAPEE HPVLLTEAPL NPKANREKMT QIMFETFNTP AMYVAIQAVL SLYASGRTTG IVMDSGDGVT HTVPIYEGYA LPHAILRLDL AGRDLTDYLM KILTERGYSF TTTAEREIVR DIKEKLCYVA LDFEQEMATA ASSSSLEKSY ELPDGQVITI GNERFRCPEA LFQPSFLGME SAGIHETTFN SIMKCDVDIR KDLYANTVLS GGTTMYPGIA DRMQKEITAL APSTMKIKII APPERKYSVW IGGSILASLS TFQQMWISKQ EYDESGPSIV HRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #2: S. pombe Cdc12

MacromoleculeName: S. pombe Cdc12 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SKDDLHKTTG LTRRPTRRLK QMHWEKLNSG LEFTFWTGPS DEANKILETL HTSGVLDELD ESFAMKEAK TLVKKTCART DYMSSELQKL FGIHFHKLSH KNPNEIIRMI LHCDDSMNEC V EFLSSDKV LNQPKLKADL EPYRIDWANG GDLVNSEKDA SELSRWDYLY ...String:
SKDDLHKTTG LTRRPTRRLK QMHWEKLNSG LEFTFWTGPS DEANKILETL HTSGVLDELD ESFAMKEAK TLVKKTCART DYMSSELQKL FGIHFHKLSH KNPNEIIRMI LHCDDSMNEC V EFLSSDKV LNQPKLKADL EPYRIDWANG GDLVNSEKDA SELSRWDYLY VRLIVDLGGY WN QRMNALK VKNIIETNYE NLVRQTKLIG RAALELRDSK VFKGLLYLIL YLGNYMNDYV RQA KGFAIG SLQRLPLIKN ANNTKSLLHI LDITIRKHFP QFDNFSPELS TVTEAAKLNI EAIE QECSE LIRGCQNLQI DCDSGALSDP TVFHPDDKIL SVILPWLMEG TKKMDFLKEH LRTMN TTLN NAMRYFGEQP NDPNSKNLFF KRVDSFIIDY SKARSDNLKS EEEEASQHRR LNLVN

UniProtKB: Cell division control protein 12

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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
100.0 mMsodium chlorideNaCl
2.0 mMmagnesium chlorideMgCl2
1.0 mMEGTA
0.5 mMTCEP

Details: 1xKMEH (10 mM HEPES pH 7.1, 100 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.5 mM TCEP)
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: 1 / Number real images: 12009 / Average electron dose: 64.3 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: 2443647 / Details: Particles picked using SPHIRE-crYOLO.
Startup modelType of model: EMDB MAP
EMDB ID:

19496


Details: Cdc12 bound to phalloidin-stabilized F-actin.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0)
Details: Final reconstruction displays preferred orientation.
Number images used: 173582
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.1.0) / Details: 3D classification in RELION.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8rtt


Chain - Source name: PDB / Chain - Initial model type: experimental model

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