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- EMDB-22401: Rubisco at apo state -

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Basic information

Entry
Database: EMDB / ID: EMD-22401
TitleRubisco at apo state
Map dataRubisco at apo state
Sample
  • Complex: Rubisco at apo state
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small chain 2, chloroplastic
  • Ligand: water
KeywordsRubisco / PLANT PROTEIN / Pyrenoid
Function / homology
Function and homology information


photorespiration / ribulose-bisphosphate carboxylase / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / chloroplast stroma / chloroplast / monooxygenase activity / magnesium ion binding
Similarity search - Function
Ribulose bisphosphate carboxylase, small subunit / Ribulose bisphosphate carboxylase small subunit, domain / Ribulose bisphosphate carboxylase, small subunit superfamily / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. / Ribulose bisphosphate carboxylase, large subunit, ferrodoxin-like N-terminal / Ribulose bisphosphate carboxylase large chain, N-terminal domain ...Ribulose bisphosphate carboxylase, small subunit / Ribulose bisphosphate carboxylase small subunit, domain / Ribulose bisphosphate carboxylase, small subunit superfamily / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. / Ribulose bisphosphate carboxylase, large subunit, ferrodoxin-like N-terminal / Ribulose bisphosphate carboxylase large chain, N-terminal domain / Ribulose bisphosphate carboxylase, large subunit, C-terminal / RuBisCO / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain superfamily / RuBisCO large subunit, N-terminal domain superfamily / Ribulose bisphosphate carboxylase large chain, catalytic domain
Similarity search - Domain/homology
Ribulose bisphosphate carboxylase large chain / Ribulose bisphosphate carboxylase large chain / Ribulose bisphosphate carboxylase small subunit, chloroplastic 2
Similarity search - Component
Biological speciesChlamydomonas reinhardtii (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsChou H / Matthies D / He S / Jonikas MC / Yu Z
Funding support United States, Singapore, United Kingdom, 9 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)IOS-1359682 United States
National Science Foundation (NSF, United States)MCB-1935444 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2-GM-119137 United States
Howard Hughes Medical Institute (HHMI)55108535 United States
Ministry of Education (MoE, Singapore)MOE2018-T2-2-059 Singapore
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S015531/1 United Kingdom
Leverhulme TrustRPG-2017-402 United Kingdom
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071574 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM007276 United States
CitationJournal: Nat Plants / Year: 2020
Title: The structural basis of Rubisco phase separation in the pyrenoid.
Authors: Shan He / Hui-Ting Chou / Doreen Matthies / Tobias Wunder / Moritz T Meyer / Nicky Atkinson / Antonio Martinez-Sanchez / Philip D Jeffrey / Sarah A Port / Weronika Patena / Guanhua He / ...Authors: Shan He / Hui-Ting Chou / Doreen Matthies / Tobias Wunder / Moritz T Meyer / Nicky Atkinson / Antonio Martinez-Sanchez / Philip D Jeffrey / Sarah A Port / Weronika Patena / Guanhua He / Vivian K Chen / Frederick M Hughson / Alistair J McCormick / Oliver Mueller-Cajar / Benjamin D Engel / Zhiheng Yu / Martin C Jonikas /
Abstract: Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO- ...Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.
History
DepositionAug 3, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jn4
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22401.png

Downloads & links

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Map

FileDownload / File: emd_22401.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRubisco at apo state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 192 pix.
= 251.52 Å		1.31 Å/pix.
x 192 pix.
= 251.52 Å		1.31 Å/pix.
x 192 pix.
= 251.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.49490726 - 0.8083171
Average (Standard dev.)0.000027628656 (±0.030038653)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 251.51999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z251.520251.520251.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.4950.8080.000

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Supplemental data

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Sample components

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Entire : Rubisco at apo state

EntireName: Rubisco at apo state
Components
  • Complex: Rubisco at apo state
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small chain 2, chloroplastic
  • Ligand: water

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Supramolecule #1: Rubisco at apo state

SupramoleculeName: Rubisco at apo state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: CC-2677 cw15 nit1-305 mt-5D

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Macromolecule #1: Ribulose bisphosphate carboxylase large chain

MacromoleculeName: Ribulose bisphosphate carboxylase large chain / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 52.607812 KDa
SequenceString: MVPQTETKAG AGFKAGVKDY RLTYYTPDYV VRDTDILAAF RMTPQPGVPP EECGAAVAAE SSTGTWTTVW TDGLTSLDRY KGRCYDIEP VPGEDNQYIA YVAYPIDLFE EGSVTNMFTS IVGNVFGFKA LRALRLEDLR IPPAYVKTFV GPPHGIQVER D KLNKYGRG ...String:
MVPQTETKAG AGFKAGVKDY RLTYYTPDYV VRDTDILAAF RMTPQPGVPP EECGAAVAAE SSTGTWTTVW TDGLTSLDRY KGRCYDIEP VPGEDNQYIA YVAYPIDLFE EGSVTNMFTS IVGNVFGFKA LRALRLEDLR IPPAYVKTFV GPPHGIQVER D KLNKYGRG LLGCTIKPKL GLSAKNYGRA VYECLRGGLD FTKDDENVNS QPFMRWRDRF LFVAEAIYKA QAETGEVKGH YL NATAGTC EEMMKRAV(SMC)A KELGVPIIMH DYLTGGFTAN TSLAIYCRDN GLLLHIHRAM HAVIDRQRNH GIHFRVLAK ALRMSGGDHL HSGTVVGKLE GEREVTLGFV DLMRDDYVEK DRSRGIYFTQ DWCSMPGVMP VASGGIHVWH MPALVEIFGD DACLQFGGG TLGHPWGNAP GAAANRVALE ACTQARNEGR DLAREGGDVI RSACKWSPEL AAACEVWKEI KFEFDTIDKL

UniProtKB: Ribulose bisphosphate carboxylase large chain

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Macromolecule #2: Ribulose bisphosphate carboxylase small chain 2, chloroplastic

MacromoleculeName: Ribulose bisphosphate carboxylase small chain 2, chloroplastic
type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 20.667959 KDa
SequenceString:
MAAVIAKSSV SAAVARPARS SVRPMAALKP AVKAAPVAAP AQANQMMVWT PVNNKMFETF SYLPPLSDEQ IAAQVDYIVA NGWIPCLEF AESDKAYVSN ESAIRFGSVS CLYYDNRYWT MWKLPMFGCR DPMQVLREIV ACTKAFPDAY VRLVAFDNQK Q VQIMGFLV QRPKSARDWQ PANKRSV

UniProtKB: Ribulose bisphosphate carboxylase small subunit, chloroplastic 2

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 232 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8
Details: 200 mM sorbitol, 50 mM HEPES, 50 mM KOAc, 2 mM Mg(OAc)2.4H2O and 1 mM CaCl2
GridModel: Quantifoil / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
Details: glow discharging for 60 seconds with a current of 15 mA in a Pelico EasiGlow system
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7462 pixel / Digitization - Frames/image: 1-60 / Number real images: 1834 / Average exposure time: 10.0 sec. / Average electron dose: 58.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 22500
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

Particle selectionNumber selected: 677071
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 491395
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 4 / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1gk8


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7jn4:
Rubisco in the apo state

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