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- EMDB-3694: In situ subtomogram average of Rubisco within the Chlamydomonas p... -

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Entry
Database: EMDB / ID: EMD-3694
TitleIn situ subtomogram average of Rubisco within the Chlamydomonas pyrenoid
Map dataIn situ subtomogram average of Rubisco holoenzymes within the pyrenoid of Chlamydomonas reinhardtii. Filtered to 16%u212B.
Sample
  • Complex: In situ Rubisco holoenzyme
    • Protein or peptide: Rubisco holoenzyme
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 16.5 Å
AuthorsCuellar LK / Schaffer M / Strauss M / Martinez-Sanchez A / Plitzko JM / Foerster F / Engel BD
CitationJournal: Cell / Year: 2017
Title: The Eukaryotic CO-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.
Authors: Elizabeth S Freeman Rosenzweig / Bin Xu / Luis Kuhn Cuellar / Antonio Martinez-Sanchez / Miroslava Schaffer / Mike Strauss / Heather N Cartwright / Pierre Ronceray / Jürgen M Plitzko / ...Authors: Elizabeth S Freeman Rosenzweig / Bin Xu / Luis Kuhn Cuellar / Antonio Martinez-Sanchez / Miroslava Schaffer / Mike Strauss / Heather N Cartwright / Pierre Ronceray / Jürgen M Plitzko / Friedrich Förster / Ned S Wingreen / Benjamin D Engel / Luke C M Mackinder / Martin C Jonikas /
Abstract: Approximately 30%-40% of global CO fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is ...Approximately 30%-40% of global CO fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.
History
DepositionApr 26, 2017-
Header (metadata) releaseMay 3, 2017-
Map releaseMay 3, 2017-
UpdateOct 4, 2017-
Current statusOct 4, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_3694.map.gz / Format: CCP4 / Size: 686.5 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ subtomogram average of Rubisco holoenzymes within the pyrenoid of Chlamydomonas reinhardtii. Filtered to 16%u212B.
Voxel sizeX=Y=Z: 3.42 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-5.7059126 - 3.9495337
Average (Standard dev.)0.000000003168763 (±1.)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions565656
Spacing565656
CellA=B=C: 191.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z565656
origin x/y/z0.0000.0000.000
length x/y/z191.520191.520191.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS565656
D min/max/mean-5.7063.9500.000

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Supplemental data

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Sample components

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Entire : In situ Rubisco holoenzyme

EntireName: In situ Rubisco holoenzyme
Components
  • Complex: In situ Rubisco holoenzyme
    • Protein or peptide: Rubisco holoenzyme

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Supramolecule #1: In situ Rubisco holoenzyme

SupramoleculeName: In situ Rubisco holoenzyme / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: In situ subtomogram average generated from Rubisco holoenzymes imaged within the native Chlamydomonas pyrenoid. Cells were thinned by focused ion beam milling.
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: mat3-4
Molecular weightTheoretical: 540 KDa

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Macromolecule #1: Rubisco holoenzyme

MacromoleculeName: Rubisco holoenzyme / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
SequenceString: MVPQTETKAG AGFKAGVKDY RLTYYTPDYV VRDTDILAAF RMTPQPGVPP EECGAAVAAE SSTGTWTTVW TDGLTSLDRY KGRCYDIEPV PGEDNQYIAY VAYPIDLFEE GSVTNMFTSI VGNVFGFKAL RALRLEDLRI PPAYVKTFVG PPHGIQVERD KLNKYGRGLL ...String:
MVPQTETKAG AGFKAGVKDY RLTYYTPDYV VRDTDILAAF RMTPQPGVPP EECGAAVAAE SSTGTWTTVW TDGLTSLDRY KGRCYDIEPV PGEDNQYIAY VAYPIDLFEE GSVTNMFTSI VGNVFGFKAL RALRLEDLRI PPAYVKTFVG PPHGIQVERD KLNKYGRGLL GCTIKPKLGL SAKNYGRAVY ECLRGGLDFT KDDENVNSQP FMRWRDRFLF VAEAIYKAQA ETGEVKGHYL NATAGTCEEM MKRAVCAKEL GVPIIMHDYL TGGFTANTSL AIYCRDNGLL LHIHRAMHAV IDRQRNHGIH FRVLAKALRM SGGDHLHSGT VVGKLEGERE VTLGFVDLMR DDYVEKDRSR GIYFTQDWCS MPGVMPVASG GIHVWHMPAL VEIFGDDACL QFGGGTLGHP WGNAPGAAAN RVALEACTQA RNEGRDLARE GGDVIRSACK WSPELAAACE VWKEIKFEFD TIDKL

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details: Blotted for 10 seconds with 10 blot force before plunging..
DetailsRubisco holoenzymes within the native Chlamydomonas pyrenoid. Whole cells were plunge-frozen onto EM grids and then thinned with a focused ion beam instrument.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 6.457 µm / Calibrated defocus min: 5.077 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Average exposure time: 1.5 sec. / Average electron dose: 1.5 e/Å2
Details: Images were collected in movie mode at 17 frames per second
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 1 / Number images used: 49828 / Reference model: PDB entry 1GK8, lowpass filtered to 33 A
Method: automated template matching and exhaustive extraction
Software:
Namedetails
PyTomtemplate matching
Amiramask segmentation

Details: Template matching hits were filtered with a manually-segmented mask, limiting subvolume extraction to the pyrenoid matrix volume.
CTF correctionSoftware - Name: IMOD / Software - details: ctfplotter, ctfphaseflip
Details: Defocus of individual tilts was estimated with IMOD's ctfplotter, and CTF correction was performed with ctfphaseflip
Final 3D classificationNumber classes: 166 / Avg.num./class: 300 / Software - Name: PyTom
Details: 46,567 true-positive subvolumes retained; 3,261 false-positive subvolumes discarded. Hierarchical clustering was used to determine which classes to discard (11 out of 166 classes discarded).
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION / Details: Angles known from tilt-series acquisition.
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 16.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION
Details: The 30,000 subvolumes with the highest template matching cross-correlation scores were used for the final reconstruction. Cumulative electron dose was restricted for the final average by ...Details: The 30,000 subvolumes with the highest template matching cross-correlation scores were used for the final reconstruction. Cumulative electron dose was restricted for the final average by only using the central 60 degrees of the tilt-series (-30 to +30). 16.5 A (FSC 0.143 cut-off) gold-standard resolution. 15.5 A (FSC 0.3 cut-off) cross-resolution of the full dataset to the crystal structure.
Number subtomograms used: 30000
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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