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- PDB-8uz1: Straight actin filament from Arp2/3 branch junction sample (ADP-BeFx) -

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Basic information

Entry
Database: PDB / ID: 8uz1
TitleStraight actin filament from Arp2/3 branch junction sample (ADP-BeFx)
ComponentsActin, alpha skeletal muscle
KeywordsCYTOSOLIC PROTEIN / actin / arp2/3 / cytoskeleton / branch
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
ADENOSINE-5'-DIPHOSPHATE / BERYLLIUM TRIFLUORIDE ION / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsChavali, S.S. / Chou, S.Z. / Sindelar, C.V.
Funding support United States, 3items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 20, 2024Group: Database references / Source and taxonomy / Category: entity_src_nat / struct_ref / struct_ref_seq
Item: _entity_src_nat.common_name / _entity_src_nat.pdbx_ncbi_taxonomy_id ..._entity_src_nat.common_name / _entity_src_nat.pdbx_ncbi_taxonomy_id / _entity_src_nat.pdbx_organism_scientific / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
J: Actin, alpha skeletal muscle
K: Actin, alpha skeletal muscle
L: Actin, alpha skeletal muscle
M: Actin, alpha skeletal muscle
N: Actin, alpha skeletal muscle
O: Actin, alpha skeletal muscle
P: Actin, alpha skeletal muscle
Q: Actin, alpha skeletal muscle
R: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)378,30536
Polymers373,6479
Non-polymers4,65827
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 41516.340 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit)
References: UniProt: P68135, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: BeF3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Arp2/3 branch complex (ADP-BeFx state)COMPLEXS. pombe arp2/3 complex (ADP-Befx state) in a branch junction with chicken skeletal actin mother and daughter filaments (ADP-BeFx state)#10MULTIPLE SOURCES
2Arp2/3 complexCOMPLEXS. pombe arp2/3 complex (ADP state)1NATURAL
3ActinORGANELLE OR CELLULAR COMPONENTActin mother and daughter filaments#11NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Schizosaccharomyces pombe (fission yeast)4896
33Gallus gallus (chicken)9031
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 ...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).
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
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.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
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 ...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.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.3 sec. / Electron dose: 51.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 8519
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.
EM imaging opticsEnergyfilter name: GIF Quantum LS
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.
Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
11cryoSPARC3final Euler assignment
12cryoSPARC3classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.74 ° / Axial rise/subunit: 27.38 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1611831
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 543085 / Algorithm: FOURIER SPACE
Details: For the final 3D reconstruction, no helical symmetry was applied
Num. of class averages: 1 / Symmetry type: HELICAL

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