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- EMDB-42829: Straight actin filament from Arp2/3 branch junction sample (ADP) -

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Basic information

Entry
Database: EMDB / ID: EMD-42829
TitleStraight actin filament from Arp2/3 branch junction sample (ADP)
Map dataHelical F-actin from Arp2/3 branch complex (ADP)
Sample
  • Organelle or cellular component: Actin
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsactin / arp2/3 / cytoskeleton / branch / CYTOSOLIC PROTEIN
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 / skeletal muscle thin filament assembly / actin filament bundle 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 / skeletal muscle thin filament assembly / actin filament bundle 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 speciesGallus gallus (chicken) / Oryctolagus cuniculus (rabbit)
Methodhelical reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsChavali SS / Chou SZ / Sindelar CV
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136656 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM026338 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM110530 United States
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structures reveal how phosphate release from Arp3 weakens actin filament branches formed by Arp2/3 complex.
Authors: Sai Shashank Chavali / Steven Z Chou / Wenxiang Cao / Thomas D Pollard / Enrique M De La Cruz / Charles V Sindelar /
Abstract: Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 ...Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeF bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeF transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of P from the ADP-P transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics.
History
DepositionNov 14, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42829.png

Downloads & links

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Map

FileDownload / File: emd_42829.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical F-actin from Arp2/3 branch complex (ADP)
Voxel sizeX=Y=Z: 1.346 Å
Density
Contour LevelBy EMDB: 0.4
Minimum - Maximum-2.21349 - 4.047427
Average (Standard dev.)0.001347616 (±0.117038645)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 344.576 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Helical F-actin from Arp2/3 branch complex (ADP)

Fileemd_42829_half_map_1.map
AnnotationHelical F-actin from Arp2/3 branch complex (ADP)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Helical F-actin from Arp2/3 branch complex (ADP)

Fileemd_42829_half_map_2.map
AnnotationHelical F-actin from Arp2/3 branch complex (ADP)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Actin

EntireName: Actin
Components
  • Organelle or cellular component: Actin
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Actin

SupramoleculeName: Actin / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Actin filament
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 9 / 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: 41.51634 KDa
SequenceString: ETTALVCDNG SGLVKAGFAG DDAPRAVFPS IVGRPRHQGV MVGMGQKDSY VGDEAQSKRG ILTLKYPIE(HIC) GIITNW DDM EKIWHHTFYN ELRVAPEEHP TLLTEAPLNP KANREKMTQI MFETFNVPAM YVAIQAVLSL YASGRTTGIV LDSGDGV TH NVPIYEGYAL ...String:
ETTALVCDNG SGLVKAGFAG DDAPRAVFPS IVGRPRHQGV MVGMGQKDSY VGDEAQSKRG ILTLKYPIE(HIC) GIITNW DDM EKIWHHTFYN ELRVAPEEHP TLLTEAPLNP KANREKMTQI MFETFNVPAM YVAIQAVLSL YASGRTTGIV LDSGDGV TH NVPIYEGYAL PHAIMRLDLA GRDLTDYLMK ILTERGYSFV TTAEREIVRD IKEKLCYVAL DFENEMATAA SSSSLEKS Y ELPDGQVITI GNERFRCPET LFQPSFIGME SAGIHETTYN SIMKCDIDIR KDLYANNVMS GGTTMYPGIA DRMQKEITA LAPSTMKIKI IAPPERKYSV WIGGSILASL STFQQMWITK QEYDEAGPSI VHRKCF

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: 9 / 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: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 9 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: The samples were incubated on the grid for 50 s and the extra solution was blotted using two Vitrobot filter papers (0.55/20 mm, Grade 595, Ted Pella) for 4 s at 0 blot force. The grids were ...Details: The samples were incubated on the grid for 50 s and the extra solution was blotted using two Vitrobot filter papers (0.55/20 mm, Grade 595, Ted Pella) for 4 s at 0 blot force. The grids were plunged into liquid ethane at ~180 degrees C with a wait time of 0.5 s..
DetailsActin monomers with bound Ca2+ were converted to Mg2+-actin by equilibrating with 50 micromolar MgCl2 and 0.2 mM EGTA (pH 7.5) for 10 min on ice. Actin was polymerized in the presence of capping protein (CP) by sequentially mixing 8.75 micromolar Mg-ATP-actin monomers with 0.75 micromolar CP and equilibrated at room temperature for 1 h. In parallel, Arp2/3 complex (0.4 micromolar) in QB buffer was activated by mixing 0.85 micromolar GCN4-VCA and 50 micromolar ATP and incubated at 4 degrees for 1 h. The capped actin filaments sample was then gently mixed with an equal volume of activated Arp2/3 complex sample using cut pipette tips and equilibrated at 4 degrees for 5 min. Daughter filaments were formed and elongated in the presence of CP by adding 0.25 micromolar Mg-actin monomers, 50 micromolar ATP, and 40 nM CP and incubated for 5 min at room temperature. This step was subsequently repeated 4 more times, then aged for ~90 min before preparing grids for cryo-EM data collection.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Details: Electron micrographs for image reconstructions were collected using Titan Krios equipped with X-cold field emission gun at 300 kV, Gatan image filter with slit width of 20 eV and a nanoprobe.
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsThe vitrified grids were screened for sample homogeneity and ice thickness in a Glacios 200 kV transmission electron microscope equipped with Gatan K2 summit camera. Electron micrographs for image reconstructions were collected using Titan Krios equipped with X-cold field emission gun at 300 kV, Gatan image filter with slit width of 20 eV and a nanoprobe.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 8519 / Average exposure time: 3.3 sec. / Average electron dose: 51.4 e/Å2
Details: Each movie contains 41 frames with a frame time of 0.08 s. A dose rate of 28.4 counts/pixel/s and a physical pixel size of 1.346 Angstroms was used.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 2320824 / Software - Name: cryoSPARC / Software - details: filament
Startup modelType of model: OTHER
Details: Low-pass filtered F-actin structure (20 A resolution)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final reconstructionApplied symmetry - Helical parameters - Δz: 27.42 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.76 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0)
Details: For the final 3D reconstruction, no helical symmetry was applied
Number images used: 490230
FSC plot (resolution estimation)

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