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- EMDB-1906: Tomographic reconstruction of unstained, frozen-hydrated thylakoi... -

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Basic information

Entry
Database: EMDB / ID: EMD-1906
TitleTomographic reconstruction of unstained, frozen-hydrated thylakoid membranes from spinach chloroplasts.
Map dataTomographic reconstruction of photosynthetic membranes from spinach chloroplasts resuspended in an isotonic buffer and imaged in an unstained frozen-hydrated state.
Sample
  • Sample: Thylakoid membrane from spinach.
  • Organelle or cellular component: Thylakoid membrane
KeywordsPhotosynthesis / thylakoid membrane / CF1-CF0 proton ATPase / photosystem / cytochrome b6f / ribosome / chloroplast.
Biological speciesSpinacia oleracea (spinach)
Methodelectron tomography / cryo EM / negative staining / Resolution: 45.0 Å
AuthorsFord RC / Holzenburg A
CitationJournal: Cystal Research and Technology / Year: 2013
Title: Organization of protein complexes and a mechanism for grana formation in photosynthetic membranes as revealed by cryo-electron microscopy
Authors: Ford RC / Holzenburg A
History
DepositionJun 8, 2011-
Header (metadata) releaseJun 20, 2011-
Map releaseJun 14, 2012-
UpdateOct 2, 2013-
Current statusOct 2, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1906.tif

Downloads & links

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Map

FileDownload / File: emd_1906.map.gz / Format: CCP4 / Size: 294.5 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationTomographic reconstruction of photosynthetic membranes from spinach chloroplasts resuspended in an isotonic buffer and imaged in an unstained frozen-hydrated state.
Voxel sizeX=Y=Z: 17.6 Å
Density
Contour LevelBy AUTHOR: 150.0
Minimum - Maximum0.0 - 255.0
Average (Standard dev.)125.452964780000002 (±16.861917500000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1-14
Dimensions991991161
Spacing991991161
CellA: 17441.6 Å / B: 17441.6 Å / C: 2833.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z17.617.617.6
M x/y/z991991161
origin x/y/z0.0000.0000.000
length x/y/z17441.60017441.6002833.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-1-14
NC/NR/NS991991161
D min/max/mean0.000255.000125.453

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Supplemental data

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Sample components

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Entire : Thylakoid membrane from spinach.

EntireName: Thylakoid membrane from spinach.
Components
  • Sample: Thylakoid membrane from spinach.
  • Organelle or cellular component: Thylakoid membrane

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Supramolecule #1000: Thylakoid membrane from spinach.

SupramoleculeName: Thylakoid membrane from spinach. / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Thylakoid membrane

SupramoleculeName: Thylakoid membrane / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Photosynthetic membrane
Details: Buffer - 20mM MES, 5mM MgCl2, 15mM NaCl, pH6.5, 150mM sorbitol. 3microlitres of membranes at 2mg per ml chlorophyll were directly applied to electron microscope grids (Quantifoil carbon ...Details: Buffer - 20mM MES, 5mM MgCl2, 15mM NaCl, pH6.5, 150mM sorbitol. 3microlitres of membranes at 2mg per ml chlorophyll were directly applied to electron microscope grids (Quantifoil carbon grids with 1.2 micron holes). After blotting excess liquid, (2x 1s blots at 95 percent relative humidity in a Vitrobot device), the grids were rapidly frozen by plunging into liquid ethane and transferred at less than 110K to the Gatan cryo-stage of the electron microscope.
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Spinacia oleracea (spinach) / synonym: Spinach / Tissue: Leaf / Organelle: Chloroplast

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography

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Sample preparation

Concentration2 mg/mL
BufferpH: 6.5
Details: 20mM MES, 5mM MgCl2, 15mM NaCl, pH6.5, 150mM sorbitol.
StainingType: NEGATIVE / Details: No stain.
GridDetails: Quantifoil 400mesh carbon grids with 1.2 micron holes.
VitrificationCryogen name: NITROGEN / Chamber humidity: 95 % / Chamber temperature: 97 K / Instrument: OTHER
Details: Vitrification instrument: Vitrobot FEI. Rapid plunging into liquid ethane.
Method: 2 x 1 sec blots before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 96 K / Max: 98 K / Average: 97 K
Alignment procedureLegacy - Astigmatism: Astigmatism corrected by live FFT.
Image recordingDigitization - Sampling interval: 15 µm / Number real images: 57 / Average electron dose: 45 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 33998 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 34000
Sample stageSpecimen holder: Single tilt / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 49 ° / Tilt series - Axis1 - Angle increment: 1 °
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Details1 degree increments above 45 deg, 2.5 degree increments below this.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 45.0 Å / Resolution method: OTHER / Software - Name: eTOMO
Details: Standard eTOMO procedures for single axis tilt tomograms. Fiducials were selected from the tomogram and tracked in each image by hand initially.
Number images used: 57
CTF correctionDetails: none

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