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- PDB-7aqk: Model of the actin filament Arp2/3 complex branch junction in cells -

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Basic information

Entry
Database: PDB / ID: 7aqk
TitleModel of the actin filament Arp2/3 complex branch junction in cells
Components
  • (Actin-related protein ...) x 7
  • Actin, alpha skeletal muscle, ACTA1
KeywordsSTRUCTURAL PROTEIN / Actin / Arp2-3 complex / Cytoskeleton
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 9 Å
AuthorsFaessler, F. / Dimchev, G. / Hodirnau, V.V. / Wan, W. / Schur, F.K.M.
Funding support Austria, 1items
OrganizationGrant numberCountry
Austrian Science FundP33367 Austria
CitationJournal: Nat Commun / Year: 2020
Title: Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction.
Authors: Florian Fäßler / Georgi Dimchev / Victor-Valentin Hodirnau / William Wan / Florian K M Schur /
Abstract: The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves ...The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 22, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 6, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
b: Actin-related protein 2, Arp2
a: Actin-related protein 3, Arp3
c: Actin-related protein 2/3 complex subunit 1b, ArpC1b
d: Actin-related protein 2/3 complex subunit 2, ArpC2
e: Actin-related protein 2/3 complex subunit 3, ArpC3
f: Actin-related protein 2/3 complex subunit 4, ArpC4
g: Actin-related protein 2/3 complex subunit 5, ArpC5
h: Actin, alpha skeletal muscle, ACTA1
i: Actin, alpha skeletal muscle, ACTA1
j: Actin, alpha skeletal muscle, ACTA1
k: Actin, alpha skeletal muscle, ACTA1
l: Actin, alpha skeletal muscle, ACTA1
m: Actin, alpha skeletal muscle, ACTA1
n: Actin, alpha skeletal muscle, ACTA1
o: Actin, alpha skeletal muscle, ACTA1
p: Actin, alpha skeletal muscle, ACTA1
q: Actin, alpha skeletal muscle, ACTA1
r: Actin, alpha skeletal muscle, ACTA1


Theoretical massNumber of molelcules
Total (without water)687,29718
Polymers687,29718
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Actin-related protein ... , 7 types, 7 molecules bacdefg

#1: Protein Actin-related protein 2, Arp2


Mass: 44818.711 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Source: (natural) Mus musculus (house mouse) / Cell line: NIH-3T3
#2: Protein Actin-related protein 3, Arp3


Mass: 47428.031 Da / Num. of mol.: 1 / Mutation: From Bos taurus to Mus Musculus I259V / Source method: isolated from a natural source
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Source: (natural) Mus musculus (house mouse) / Cell line: NIH-3T3
#3: Protein Actin-related protein 2/3 complex subunit 1b, ArpC1b


Mass: 41016.738 Da / Num. of mol.: 1
Mutation: From Bos taurus to Mus Musculus V43N, Q44K, V58I, D63E, K109N, S154N, N213S, A229V, S256N, S274N, G277V, K278T, L296M, S313G, A216T, R360K
Source method: isolated from a natural source
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Source: (natural) Mus musculus (house mouse) / Cell line: NIH-3T3
#4: Protein Actin-related protein 2/3 complex subunit 2, ArpC2


Mass: 34402.043 Da / Num. of mol.: 1 / Mutation: From Bos taurus to Mus Musculus Y84F, S90P / Source method: isolated from a natural source
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Source: (natural) Mus musculus (house mouse) / Cell line: NIH-3T3
#5: Protein Actin-related protein 2/3 complex subunit 3, ArpC3


Mass: 20572.666 Da / Num. of mol.: 1 / Mutation: From Bos taurus to Mus Musculus I24L / Source method: isolated from a natural source
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Source: (natural) Mus musculus (house mouse) / Cell line: NIH-3T3
#6: Protein Actin-related protein 2/3 complex subunit 4, ArpC4


Mass: 19697.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Source: (natural) Mus musculus (house mouse) / Cell line: NIH-3T3
#7: Protein Actin-related protein 2/3 complex subunit 5, ArpC5


Mass: 16295.317 Da / Num. of mol.: 1 / Mutation: From Bos taurus to Mus Musculus D28E / Source method: isolated from a natural source
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Source: (natural) Mus musculus (house mouse) / Cell line: NIH-3T3

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Protein , 1 types, 11 molecules hijklmnopqr

#8: Protein
Actin, alpha skeletal muscle, ACTA1 /


Mass: 42096.953 Da / Num. of mol.: 11 / Source method: isolated from a natural source
Details: As models derived from Oryctolagus cuniculus actin have been used for fitting, also the Oryctolagus cuniculus sequence (as well as the corresponding UniProt identifier) is given here, even ...Details: As models derived from Oryctolagus cuniculus actin have been used for fitting, also the Oryctolagus cuniculus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Source: (natural) Mus musculus (house mouse) / Cell line: NIH-3T3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Actin Filament Arp2/3 Complex Branch Junction / Type: CELL
Details: Structure obtained from the actin network of extracted and fixed mouse fibroblast lamellipodia
Entity ID: #1-#8 / Source: NATURAL
Source (natural)Organism: Mus musculus (house mouse) / Cellular location: Lamellipodium
Buffer solutionpH: 6.1 / Details: Adjust to pH 6.1 using NaOH
Buffer component
IDConc.NameBuffer-ID
110 mMMES1
2150 mMsodium chloride1
35 mMEGTA1
45 mMGlucose1
55 mMMagnesium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: After glow discharging of the grid and prior to the seeding of cells, the grid was coated using 25ug/ml Fibronectin
Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K
Details: Leica GP2, 3,5sec back-blotting, sensor on, 0,1mm movement after contact, manually pre-blotted within the chamber prior to the application of fiducials

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 42000 X / Nominal defocus max: -5.5 nm / Nominal defocus min: -1.75 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.21 sec. / Electron dose: 2.79 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
Details: Images were collected in movie-mode at 7 frames per tilt
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Dynamo1.133volume selection
2Warp1.07volume selection
3SerialEMimage acquisition
5Warp1.07CTF correction
8UCSF Chimera1.13.1model fitting
11RELION3.0.9final Euler assignment
12RELION3.0.9classification
13RELION3.0.93D reconstruction
14ISOLDE1.0b5model refinement
15Coot0.8.9.3model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14296
Details: Final reconstruction in RELION was performed after Multiple particle refinement in M version 1.0.9.
Num. of class averages: 1 / Symmetry type: POINT
EM volume selectionMethod: Template Matching
Details: After first classification in Dynamo and re-extraction in Warp 17,146 subvolumes remained.
Num. of tomograms: 131 / Num. of volumes extracted: 39300
Reference model: Reference generated from manually selected particles
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-IDPdb chain residue range
11TYQA12-39
21TYQA152-353
31TYQA1360-418
41TYQB1151-345
51TYQC11-288
61TYQC1319-372
71TYQD11-208
81TYQD1217-281
91TYQE12-150
101TYQE1155-174
111TYQF13-168
121TYQG136-151
134JD2B14-150
144JD2B1346-387
151K8KC1297-309
166T20A15-375
176T20B15-375
186T20C15-375
196T20D15-375
206T20E15-375

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