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- EMDB-43893: Structure of the auto-fluorescent membrane-bound red body organel... -

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Basic information

Entry
Database: EMDB / ID: EMD-43893
TitleStructure of the auto-fluorescent membrane-bound red body organelle from Nannochloropsis oceanica in situ
Map dataTomographic reconstruction of a cryo-lamella through a Nannochloropsis oceanica cell showing a red body within its cellular context.
Sample
  • Cell: Nannochloropsis oceanica CCMP1779
KeywordsOrganelle / Algae / Intracellular Transport / Carotenoid / LIPID TRANSPORT
Biological speciesNannochloropsis oceanica (eukaryote)
Methodelectron tomography / cryo EM
AuthorsGrob P / Danielle J / Gee CW
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Science Foundation (NSF, United States)DGE 1106400 United States
CitationJournal: To Be Published
Title: A proposed function for the red body of Nannochloropsis in the formation of the recalcitrant cell wall polymer, algaenan
Authors: Gee CW / Andersen-Ranberg J
History
DepositionFeb 29, 2024-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43893.png

Downloads & links

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Map

FileDownload / File: emd_43893.map.gz / Format: CCP4 / Size: 558.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomographic reconstruction of a cryo-lamella through a Nannochloropsis oceanica cell showing a red body within its cellular context.
Voxel sizeX=Y=Z: 22.13 Å
Density
Minimum - Maximum-0.35291177 - 0.42991072
Average (Standard dev.)0.022530546 (±0.046284698)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin53947-77
Dimensions809953190
Spacing953809190
CellA: 21089.889 Å / B: 17903.17 Å / C: 4204.6997 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Nannochloropsis oceanica CCMP1779

EntireName: Nannochloropsis oceanica CCMP1779
Components
  • Cell: Nannochloropsis oceanica CCMP1779

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Supramolecule #1: Nannochloropsis oceanica CCMP1779

SupramoleculeName: Nannochloropsis oceanica CCMP1779 / type: cell / ID: 1 / Parent: 0
Details: Tomographic reconstruction of a red body organelle from Nannochloropsis oceanica in situ
Source (natural)Organism: Nannochloropsis oceanica (eukaryote) / Strain: CCMP1779

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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: 8.1
Component:
ConcentrationFormulaName
2.0 mMNaH4Clammonium chloride
0.083 mMNaH2PO4monosodium phosphate
10.0 mMTRIS-HClTrisTRIS hydrochloride
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 12 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.027 kPa / Details: the grid was soaked in chloroform before use
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: Blotted manually from opposite side of the grid.
DetailsNannochloropsis oceanica CCMP1779 cells were grown in artificial seawater and f-media enrichment entrained to a 12-12 light-dark photoperiod and sampled in mid-log phase (~1x10^7 cells/ml), shortly after subjective dark when cells are undergoing division.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.037 / Focused ion beam - Duration: 600 / Focused ion beam - Temperature: 123 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 380
Focused ion beam - Details: Grids were transfered to a Leica Ace 900 (Leica Microsystems) for coating with 5nm of platinum prior to being transferred to the Zeiss Crossbeam 540 (Zeiss, Germany). The ...Focused ion beam - Details: Grids were transfered to a Leica Ace 900 (Leica Microsystems) for coating with 5nm of platinum prior to being transferred to the Zeiss Crossbeam 540 (Zeiss, Germany). The Zeiss Crossbeam 540 operated with a Leica CryoStage (Leica Microsystems, GmbH) cooled to -150 deg. C was used for milling and imaging of the frozen cells. To create lamellae used for cryo-tomography, milling was performed with a gallium ion source at an energy of 37 pA and a working distance of 5mm. Imaging of the grid and milled lamellae was done using an Everhart-Thornley detector at 2.0 kV.. The value given for _em_focused_ion_beam.instrument is Zeiss Crossbeam 540. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 15000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 35 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 100.0 e/Å2
Details: images were collected in movie mode and super resolution
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.11.24) / Software - details: Etomo / Number images used: 61
Detailsthe movie frames were aligned with MotionCor2 and binned to normal resolution

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