EMDB-42830: Straight actin filament from Arp2/3 branch junction sample (ADP-BeFx)

Basic information

Entry

Database: EMDB / ID: EMD-42830

• Title: Straight actin filament from Arp2/3 branch junction sample (ADP-BeFx)
• Map data: cryosparc final reconstruction, sharpened

Sample

• Complex: Arp2/3 branch complex (ADP-BeFx state)
  • Complex: Arp2/3 complex
  • Organelle or cellular component: Actin
    • Protein or peptide: Actin, alpha skeletal muscle
• Ligand: ADENOSINE-5'-DIPHOSPHATE
• Ligand: MAGNESIUM ION
• Ligand: BERYLLIUM TRIFLUORIDE ION

• Keywords: actin / arp2/3 / cytoskeleton / branch / CYTOSOLIC PROTEIN

Function / homology

Function:

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

Domain/homology:

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

Component:

Actin, alpha skeletal muscle

• Biological species: Schizosaccharomyces pombe (fission yeast) / Gallus gallus (chicken) / Oryctolagus cuniculus (rabbit)
• Method: helical reconstruction / cryo EM / Resolution: 3.6 Å
• Authors: Chavali SS / Chou SZ / Sindelar CV

Funding support

United States, 3 items

Organization	Grant number	Country
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

• Citation: Journal: 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 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

Deposition	Nov 14, 2023	-
Header (metadata) release	Jan 31, 2024	-
Map release	Jan 31, 2024	-
Update	Mar 20, 2024	-
Current status	Mar 20, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_42830.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: cryosparc final reconstruction, sharpened
• Voxel size: X=Y=Z: 1.346 Å

Density

Contour Level	By AUTHOR: 0.3
Minimum - Maximum	-1.0769811 - 1.989544
Average (Standard dev.)	0.001641151 (±0.06225886)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 344.576 Å α=β=γ: 90.0 °

Supplemental data

Additional map: cryosparc final reconstruction, no sharpening

• File: emd_42830_additional_1.map
• Annotation: cryosparc final reconstruction, no sharpening

Half map: cryosparc first half-map reconstruction

• File: emd_42830_half_map_1.map
• Annotation: cryosparc first half-map reconstruction

Half map: cryosparc second half-map reconstruction

• File: emd_42830_half_map_2.map
• Annotation: cryosparc second half-map reconstruction

Sample components

Entire : Arp2/3 branch complex (ADP-BeFx state)

• Entire: Name: Arp2/3 branch complex (ADP-BeFx state)

Components

• Complex: Arp2/3 branch complex (ADP-BeFx state)
  • Complex: Arp2/3 complex
  • Organelle or cellular component: Actin
    • Protein or peptide: Actin, alpha skeletal muscle
• Ligand: ADENOSINE-5'-DIPHOSPHATE
• Ligand: MAGNESIUM ION
• Ligand: BERYLLIUM TRIFLUORIDE ION

Supramolecule #1: Arp2/3 branch complex (ADP-BeFx state)

• Supramolecule: Name: Arp2/3 branch complex (ADP-BeFx state) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1; Details: S. pombe arp2/3 complex (ADP-Befx state) in a branch junction with chicken skeletal actin mother and daughter filaments (ADP-BeFx state)

Supramolecule #2: Arp2/3 complex

• Supramolecule: Name: Arp2/3 complex / type: complex / ID: 2 / Parent: 1 / Details: S. pombe arp2/3 complex (ADP state)
• Source (natural): Organism: Schizosaccharomyces pombe (fission yeast)

Supramolecule #3: Actin

• Supramolecule: Name: Actin / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #1 / Details: Actin mother and daughter filaments
• Source (natural): Organism: Gallus gallus (chicken)

Macromolecule #1: Actin, alpha skeletal muscle

• Macromolecule: Name: 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 weight: Theoretical: 41.51634 KDa

Sequence

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

Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 9 / Formula: ADP
• Molecular weight: Theoretical: 427.201 Da

Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

Macromolecule #3: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 9 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Macromolecule #4: BERYLLIUM TRIFLUORIDE ION

• Macromolecule: Name: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 4 / Number of copies: 9 / Formula: BEF
• Molecular weight: Theoretical: 66.007 Da

Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

Experimental details

Structure determination

• Method: cryo EM
• Processing: helical reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7.5
• Vitrification: Cryogen 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 plunged into liquid ethane at ~180 degrees C with a wait time of 0.5 s..
• Details: Actin 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. A final concentration of 2 mM BeFx (prepared using 2 mM BeSO4 and 10 mM NaF) was added during the 4th step of daughter filament formation (as mentioned above).

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination 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 optics: Energy 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 stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Details: The 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 recording: Film or detector model: GATAN K3 (6k x 4k) / Number real images: 8519 / Average exposure time: 3.3 sec. / Average electron dose: 51.4 e/Ų; 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

Image processing

• Segment selection: Number selected: 1611831
• Startup model: Type of model: NONE; Details: These particles were subjected to 3-dimensional structure refinement and classification using 'Ab-initio 3D reconstruction' (10 classes)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
• Final reconstruction: Number classes used: 1 ; Applied symmetry - Helical parameters - Δz: 27.38 Å; Applied symmetry - Helical parameters - Δ&Phi: -166.74 °; Applied symmetry - Helical parameters - Axial symmetry: C₁ (asymmetric); Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 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: 543085