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- EMDB-20828: Cryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol -

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Basic information

Entry
Database: EMDB / ID: EMD-20828
TitleCryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol
Map dataCryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol
Sample
  • Cell: Cryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsWatanabe R / Villa E
CitationJournal: Cell / Year: 2020
Title: The In Situ Structure of Parkinson's Disease-Linked LRRK2.
Authors: Reika Watanabe / Robert Buschauer / Jan Böhning / Martina Audagnotto / Keren Lasker / Tsan-Wen Lu / Daniela Boassa / Susan Taylor / Elizabeth Villa /
Abstract: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light ...Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.
History
DepositionOct 14, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseAug 19, 2020-
UpdateOct 7, 2020-
Current statusOct 7, 2020Processing site: RCSB / 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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Supplemental images

emd_20828.png

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Map

FileDownload / File: emd_20828.map.gz / Format: CCP4 / Size: 158.9 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationCryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol
Voxel sizeX=Y=Z: 10.8 Å
Density
Minimum - Maximum-128 - 127
Average (Standard dev.)5.5751357 (±21.909538)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1920115
Dimensions928960187
Spacing960928187
CellA: 10368.0 Å / B: 10022.4 Å / C: 2019.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z10.810.810.8
M x/y/z960928187
origin x/y/z0.0000.0000.000
length x/y/z10368.00010022.4002019.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ162182277
MAP C/R/S123
start NC/NR/NS20-19115
NC/NR/NS960928187
D min/max/mean-128.000127.0005.575

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Supplemental data

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Sample components

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Entire : Cryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol

EntireName: Cryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol
Components
  • Cell: Cryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol

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Supramolecule #1: Cryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol

SupramoleculeName: Cryo-electron tomogram of FIB-milled HEK293 cells treated with Taxol
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Cell: HEK293

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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: 7
GridDetails: unspecified
VitrificationCryogen name: ETHANE-PROPANE
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.001 nA / Focused ion beam - Duration: 50 sec. / Focused ion beam - Temperature: 70 K / Focused ion beam - Initial thickness: 4000 nm / Focused ion beam - Final thickness: 80 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is Aquilos. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 3.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 39

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