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- EMDB-50507: Structure of the undecorated pointed end of F-actin -

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Basic information

Entry
Database: EMDB / ID: EMD-50507
TitleStructure of the undecorated pointed end of F-actin
Map dataSharpened cryo-EM density map of the undecorated pointed end of actin filaments.
Sample
  • Complex: Complex of actin subunits that form the pointed end of actin filaments.
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsactin / filament / pointed end / STRUCTURAL PROTEIN
Function / homology
Function and homology information


cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament ...cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / stress fiber / skeletal muscle fiber development / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
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
Similarity search - Domain/homology
Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsBoiero Sanders M / Oosterheert W / Hofnagel O / Bieling P / Raunser S
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: Nat Commun / Year: 2024
Title: Phalloidin and DNase I-bound F-actin pointed end structures reveal principles of filament stabilization and disassembly.
Authors: Micaela Boiero Sanders / Wout Oosterheert / Oliver Hofnagel / Peter Bieling / Stefan Raunser /
Abstract: Actin filament turnover involves subunits binding to and dissociating from the filament ends, with the pointed end being the primary site of filament disassembly. Several molecules modulate filament ...Actin filament turnover involves subunits binding to and dissociating from the filament ends, with the pointed end being the primary site of filament disassembly. Several molecules modulate filament turnover, but the underlying mechanisms remain incompletely understood. Here, we present three cryo-EM structures of the F-actin pointed end in the presence and absence of phalloidin or DNase I. The two terminal subunits at the undecorated pointed end adopt a twisted conformation. Phalloidin can still bind and bridge these subunits, inducing a conformational shift to a flattened, F-actin-like state. This explains how phalloidin prevents depolymerization at the pointed end. Interestingly, two DNase I molecules simultaneously bind to the phalloidin-stabilized pointed end. In the absence of phalloidin, DNase I binding would disrupt the terminal actin subunit packing, resulting in filament disassembly. Our findings uncover molecular principles of pointed end regulation and provide structural insights into the kinetic asymmetry between the actin filament ends.
History
DepositionMay 31, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50507.png

Downloads & links

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Map

FileDownload / File: emd_50507.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM density map of the undecorated pointed end of actin filaments.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 360 pix.
= 324. Å		0.9 Å/pix.
x 360 pix.
= 324. Å		0.9 Å/pix.
x 360 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.689
Minimum - Maximum-2.9052396 - 5.176197
Average (Standard dev.)0.0022858332 (±0.09661965)
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_50507_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened cryo-EM density map of the undecorated pointed...

Fileemd_50507_additional_1.map
AnnotationUnsharpened cryo-EM density map of the undecorated pointed 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 1 of the undecorated pointed...

Fileemd_50507_half_map_1.map
AnnotationUnfiltered half map 1 of the undecorated pointed 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 undecorated pointed...

Fileemd_50507_half_map_2.map
AnnotationUnfiltered half map 2 of the undecorated pointed 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 actin subunits that form the pointed end of actin fila...

EntireName: Complex of actin subunits that form the pointed end of actin filaments.
Components
  • Complex: Complex of actin subunits that form the pointed end of actin filaments.
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of actin subunits that form the pointed end of actin fila...

SupramoleculeName: Complex of actin subunits that form the pointed end of actin filaments.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Alpha actin was purified from skeletal muscle. Actin filaments were polymerized in the presence of the formin INF2, which was purified separetely and added during polymerization prior to ...Details: Alpha actin was purified from skeletal muscle. Actin filaments were polymerized in the presence of the formin INF2, which was purified separetely and added during polymerization prior to cryo-EM grid preparation.
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Skeletal muscle

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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: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 4 / 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 #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 4 / 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
12.0 mMHEPES
100.0 mMsodium chlorideNaCl
2.1 mMmagnesium chlorideMgCl2
1.0 mMEGTA
1.0 mMTCEP
0.2 mMATP

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: CELLULOSE ACETATE / 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: 300 kV Titan Krios G3 microscope (Thermo Fisher Scientific).
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV / Details: Gatan energy filter
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
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: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 105000
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: 1935707 / Details: Particles picked using SPHIRE-crYOLO.
Startup modelType of model: EMDB MAP
EMDB ID:

3835

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 206373
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.2.1)
Details: Heterogeneous refinement in cryoSPARC at the beginning and clean up by 2D classification.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8a2t


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsReal Space Refinement in Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9fjo:
Structure of the undecorated pointed end of F-actin

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