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- EMDB-15108: Cryo-EM structure of F-actin in the Ca2+-ADP-Pi nucleotide state. -

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Basic information

Entry
Database: EMDB / ID: EMD-15108
TitleCryo-EM structure of F-actin in the Ca2+-ADP-Pi nucleotide state.
Map dataSharpened, local-resolution filtered cryo-EM density map of F-actin in the Ca2 -ADP-Pi nucleotide state.
Sample
  • Complex: rabbit skeletal alpha-actin in the filamentous state.
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: CALCIUM IONCalcium
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: water
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament ...cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / 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) / rabbit (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.15 Å
AuthorsOosterheert W / Klink BU / Belyy A / Pospich S / Raunser S
Funding supportEuropean Union, Germany, 4 items
OrganizationGrant numberCountry
European Research Council (ERC)856118European Union
Alexander von Humboldt Foundation Germany
European Molecular Biology Organization (EMBO)European Union
Max Planck Society Germany
CitationJournal: Nature / Year: 2022
Title: Structural basis of actin filament assembly and aging.
Authors: Wout Oosterheert / Björn U Klink / Alexander Belyy / Sabrina Pospich / Stefan Raunser /
Abstract: The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state. It remains unclear how F-actin hydrolyses ATP and ...The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state. It remains unclear how F-actin hydrolyses ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here we present cryo-electron microscopy structures of F-actin in all nucleotide states, polymerized in the presence of Mg or Ca at approximately 2.2 Å resolution. The structures show that actin polymerization induces the relocation of water molecules in the nucleotide-binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (P) is closed in all structures, indicating that P release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and P release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of water molecules in the nucleotide-binding pocket explain why Ca-actin shows slower polymerization rates than Mg-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.
History
DepositionJun 6, 2022-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15108.png

Downloads & links

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Map

FileDownload / File: emd_15108.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened, local-resolution filtered cryo-EM density map of F-actin in the Ca2 -ADP-Pi nucleotide state.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.7 Å/pix.
x 384 pix.
= 266.88 Å		0.7 Å/pix.
x 384 pix.
= 266.88 Å		0.7 Å/pix.
x 384 pix.
= 266.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.695 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.042
Minimum - Maximum-0.19395174 - 0.43756098
Average (Standard dev.)9.408078e-05 (±0.008236304)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 266.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15108_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 F-actin in...

Fileemd_15108_additional_1.map
Annotation3D-refined, unsharpened cryo-EM density map of F-actin in the Ca2 -ADP-Pi nucleotide state.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 1 of F-actin in the Ca2 -ADP-Pi nucleotide state.

Fileemd_15108_half_map_1.map
AnnotationUnfiltered half map 1 of F-actin in the Ca2 -ADP-Pi nucleotide state.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 2 of F-actin in the Ca2 -ADP-Pi nucleotide state.

Fileemd_15108_half_map_2.map
AnnotationUnfiltered half map 2 of F-actin in the Ca2 -ADP-Pi nucleotide state.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : rabbit skeletal alpha-actin in the filamentous state.

EntireName: rabbit skeletal alpha-actin in the filamentous state.
Components
  • Complex: rabbit skeletal alpha-actin in the filamentous state.
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: CALCIUM IONCalcium
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: water

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Supramolecule #1: rabbit skeletal alpha-actin in the filamentous state.

SupramoleculeName: rabbit skeletal alpha-actin in the filamentous state. / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The helical rise of F-actin is 27.5 Angstrom, with a helical twist of ~166.5 degrees.
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Organ: skeletal muscle / Tissue: purified from muscle acetone powder.
Molecular weightTheoretical: 15.2 kDa/nm

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: rabbit (rabbit) / Organ: 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

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

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

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 4 / Number of copies: 5 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 528 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.67 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
5.0 mMTris
50.0 mMpotassium chlorideKCl
2.0 mMcalcium chlorideCaCl2
2.0 mMsodium azideNaN3
1.0 mMdithiothreitol
50.0 mMpotassium phosphate

Details: F-phosphate buffer: 5 mM Tris, 100 mM KCl, 2 mM CaCl2, 2 mM NaN3, 1 mM DTT, 50 mM potassium phosphate pH 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV
Details: The Vitrobot was operated at 13 degrees celsius and the samples were blotted for 9 seconds with a blot force of -25..

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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: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Slit width: 15 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 10156 / Average exposure time: 3.0 sec. / Average electron dose: 88.8 e/Å2 / Details: Images were collected in supperresolution mode.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3031270
Details: We picked helical segments with a box distance of 40 pixels or 27.8 Angstrom and a minimum number of six boxes per filament.
CTF correctionSoftware - Name: CTFFIND (ver. 4.13)
Startup modelType of model: EMDB MAP
EMDB ID:

11787

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.4)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 2171987
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7ahn

DetailsThe structures were then refined through a similar protocol of iterative cycles in Coot and phenix real-space refine. All solvent molecules (ions, waters) were placed manually in Coot in the central actin subunit, and were then placed in the other subunits using NCS. Because the local resolution of each F-actin reconstruction is highest in the center and lower at the periphery of the map, we inspected all waters in each structure manually before the final phenix refinement; water molecular with poor corresponding cryo-EM density were removed.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8a2y:
Cryo-EM structure of F-actin in the Ca2+-ADP-Pi nucleotide state.

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