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- EMDB-3100: Cryo-electron tomography of a Golgi intracisternal protein array -

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Basic information

Entry
Database: EMDB / ID: EMD-3100
TitleCryo-electron tomography of a Golgi intracisternal protein array
Map dataSymmetrized average of a trans-Golgi intracisternal protein array
Sample
  • Sample: Unknown protein complex linking the cisternal membranes of the trans-Golgi
  • Organelle or cellular component: trans-Golgi intracisternal protein array
KeywordsGolgi / transmembrane protein / cisterna / focused ion beam / tomography
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 27.0 Å
AuthorsEngel BD / Schaffer M / Albert S / Asano S / Plitzko JM / Baumeister W
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: In situ structural analysis of Golgi intracisternal protein arrays.
Authors: Benjamin D Engel / Miroslava Schaffer / Sahradha Albert / Shoh Asano / Jürgen M Plitzko / Wolfgang Baumeister /
Abstract: We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo- ...We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo-electron tomography. These tomograms revealed structures within the Golgi cisternae that have not been seen before. Narrow trans-Golgi lumina were spanned by asymmetric membrane-associated protein arrays that had ∼6-nm lateral periodicity. Subtomogram averaging showed that the arrays may determine the narrow central spacing of the trans-Golgi cisternae through zipper-like interactions, thereby forcing cargo to the trans-Golgi periphery. Additionally, we observed dense granular aggregates within cisternae and intracisternal filament bundles associated with trans-Golgi buds. These native in situ structures provide new molecular insights into Golgi architecture and function.
History
DepositionJul 16, 2015-
Header (metadata) releaseAug 5, 2015-
Map releaseSep 9, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.531
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.531
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3100.png

emd_3100.png

Downloads & links

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Map

FileDownload / File: emd_3100.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSymmetrized average of a trans-Golgi intracisternal protein array
Voxel sizeX=Y=Z: 6.84 Å
Density
Contour LevelBy AUTHOR: 0.531 / Movie #1: 0.531
Minimum - Maximum-1.96310174 - 0.92108095
Average (Standard dev.)0.0 (±0.9999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 656.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.846.846.84
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z656.640656.640656.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-21-120
NX/NY/NZ432573
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-1.9630.9210.000

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Supplemental data

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Sample components

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Entire : Unknown protein complex linking the cisternal membranes of the tr...

EntireName: Unknown protein complex linking the cisternal membranes of the trans-Golgi
Components
  • Sample: Unknown protein complex linking the cisternal membranes of the trans-Golgi
  • Organelle or cellular component: trans-Golgi intracisternal protein array

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Supramolecule #1000: Unknown protein complex linking the cisternal membranes of the tr...

SupramoleculeName: Unknown protein complex linking the cisternal membranes of the trans-Golgi
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: trans-Golgi intracisternal protein array

SupramoleculeName: trans-Golgi intracisternal protein array / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Organelle: Golgi / Location in cell: trans-Golgi cisternae

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV
Details: Focused ion-beam milling was used to thin the sample

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 14600 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: -0.006 µm / Nominal defocus min: -0.005 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: Gatan
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
DateFeb 20, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 60 / Average electron dose: 60 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each tilt
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 27.0 Å / Resolution method: OTHER / Software - Name: IMOD, TOM, pyTOM / Number subtomograms used: 244
Detailstranslational symmetry was applied

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