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- EMDB-11870: Tomogram of the actin network in an extracted and fixed mouse fib... -

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Basic information

Entry
Database: EMDB / ID: EMD-11870
TitleTomogram of the actin network in an extracted and fixed mouse fibroblast lamellipodium.
Map dataSample tomogram of an extracted and fixed mouse fibroblast lamellipodium.
Sample
  • Cell: Actin network in a mouse fibroblast lamellipodium
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsFaessler F / Dimchev G / Hodirnau VV / Wan W / Schur FKM
Funding support Austria, 1 items
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.
History
DepositionOct 22, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateJan 13, 2021-
Current statusJan 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_11870.png

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Map

FileDownload / File: emd_11870.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationSample tomogram of an extracted and fixed mouse fibroblast lamellipodium.
Voxel sizeX=Y=Z: 17.096 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)22.817268 (±6.195656)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin49-4935
Dimensions720512150
Spacing512720150
CellA: 8753.152 Å / B: 12309.12 Å / C: 2564.4001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z17.09617.09617.096
M x/y/z512720150
origin x/y/z0.0000.0000.000
length x/y/z8753.15212309.1202564.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ540540540
MAP C/R/S123
start NC/NR/NS-494935
NC/NR/NS512720150
D min/max/mean-128.000127.00022.817

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Supplemental data

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Sample components

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Entire : Actin network in a mouse fibroblast lamellipodium

EntireName: Actin network in a mouse fibroblast lamellipodium
Components
  • Cell: Actin network in a mouse fibroblast lamellipodium

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Supramolecule #1: Actin network in a mouse fibroblast lamellipodium

SupramoleculeName: Actin network in a mouse fibroblast lamellipodium / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: Branched actin network of extracted and fixed mouse fibroblast lamellipodium
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 6.1
Component:
ConcentrationName
10.0 mMMES
150.0 mMsodium chloride
5.0 mMEGTA
5.0 mMGlucose
5.0 mMMagnesium chloride

Details: Adjust to pH 6.1 using NaOH
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: After glow discharging of the grid and prior to the seeding of cells, the grid was coated using 25ug/ml Fibronectin
VitrificationCryogen name: ETHANE / Chamber 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.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: AURION / Diameter: 10 nm

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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: -0.0055 µm / Nominal defocus min: -0.00175 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 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) / Number real images: 61 / Average exposure time: 1.21 sec. / Average electron dose: 2.79 e/Å2
Details: Images were collected in movie-mode with 7 frames per tilt
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.9.12) / Number images used: 61

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