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- EMDB-12325: In situ cryo-electron tomogram of the Synechocystis F4E VIPP1 mut... -

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Basic information

Entry
Database: EMDB / ID: EMD-12325
TitleIn situ cryo-electron tomogram of the Synechocystis F4E VIPP1 mutant grown in high light
Map dataContrast-enhanced tomogram by deconvolution using the TOM software toolbox (TOMdeconv). This is a binned tomogram at 4x the original pixel size.
Sample
  • Cell: Synechocystis F4E VIPP1 mutant grown in high light
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodelectron tomography / cryo EM
AuthorsWietrzynski W / Klumpe S / Heinz S / Spaniol B / Schaffer M / Rast A / Nickelsen J / Schroda M / Engel BD
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)EN 1194/1-1 (FOR2092) Germany
CitationJournal: Cell / Year: 2021
Title: Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.
Authors: Tilak Kumar Gupta / Sven Klumpe / Karin Gries / Steffen Heinz / Wojciech Wietrzynski / Norikazu Ohnishi / Justus Niemeyer / Benjamin Spaniol / Miroslava Schaffer / Anna Rast / Matthias ...Authors: Tilak Kumar Gupta / Sven Klumpe / Karin Gries / Steffen Heinz / Wojciech Wietrzynski / Norikazu Ohnishi / Justus Niemeyer / Benjamin Spaniol / Miroslava Schaffer / Anna Rast / Matthias Ostermeier / Mike Strauss / Jürgen M Plitzko / Wolfgang Baumeister / Till Rudack / Wataru Sakamoto / Jörg Nickelsen / Jan M Schuller / Michael Schroda / Benjamin D Engel /
Abstract: Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its ...Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.
History
DepositionFeb 10, 2021-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateJul 21, 2021-
Current statusJul 21, 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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Supplemental images

emd_12325.png

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Map

FileDownload / File: emd_12325.map.gz / Format: CCP4 / Size: 762.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationContrast-enhanced tomogram by deconvolution using the TOM software toolbox (TOMdeconv). This is a binned tomogram at 4x the original pixel size.
Voxel sizeX=Y=Z: 14.08 Å
Density
Minimum - Maximum-5742.0 - 2523.0
Average (Standard dev.)119.04235 (±205.15173)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions928928464
Spacing928928464
CellA: 13066.24 Å / B: 13066.24 Å / C: 6533.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z14.0814.0814.08
M x/y/z928928464
origin x/y/z0.0000.0000.000
length x/y/z13066.24013066.2406533.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS928928464
D min/max/mean-5742.0002523.000119.042

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Supplemental data

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Additional map: Original dose-filtered tomogram reconstruction. This is a binned...

Fileemd_12325_additional_1.map
AnnotationOriginal dose-filtered tomogram reconstruction. This is a binned tomogram at 4x the original pixel size.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Synechocystis F4E VIPP1 mutant grown in high light

EntireName: Synechocystis F4E VIPP1 mutant grown in high light
Components
  • Cell: Synechocystis F4E VIPP1 mutant grown in high light

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Supramolecule #1: Synechocystis F4E VIPP1 mutant grown in high light

SupramoleculeName: Synechocystis F4E VIPP1 mutant grown in high light / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)

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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
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 1800 sec. / Focused ion beam - Temperature: 91 K / Focused ion beam - Initial thickness: 3000 nm / Focused ion beam - Final thickness: 200 nm
Focused ion beam - Details: See https://bio-protocol.org/e1575 for detailed procedure.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Aquilos FIB. This is not in a list of ...Focused ion beam - Details: See https://bio-protocol.org/e1575 for detailed procedure.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Aquilos FIB. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsDose-symmetric tilt-series were acquired (Hagen et al., 2017), starting at +10 degrees to match the pre-tilt of the lamella (i.e. from -50 to +70 degrees).
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 1.0 sec. / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.5 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 61
CTF correctionSoftware - Name: IMOD

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