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- EMDB-22308: EPYC1(49-72)-bound Rubisco -

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Basic information

Entry
Database: EMDB / ID: EMD-22308
TitleEPYC1(49-72)-bound Rubisco
Map dataEPYC1(49-72) peptide-bound Rubisco. The map was processed at super resolution pixel size
Sample
  • Complex: EPYC1(49-72) peptide-bound Rubisco
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small chain 2, chloroplastic
    • Protein or peptide: LCI5
  • Ligand: water
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 small subunit, chloroplastic 2 / LCI5
Similarity search - Component
Biological speciesChlamydomonas reinhardtii (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.13 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 Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2-GM-119137 United States
National Science Foundation (NSF, United States)MCB-1935444 United States
Ministry of Education (MoE, Singapore)MOE2018-T2-2-059 Singapore
Howard Hughes Medical Institute (HHMI)55108535 United States
Leverhulme TrustRPG-2017-402 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S015531/1 United Kingdom
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM007276 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071574 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
DepositionJul 17, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateDec 23, 2020-
Current statusDec 23, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jfo
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22308.png

Downloads & links

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Map

FileDownload / File: emd_22308.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEPYC1(49-72) peptide-bound Rubisco. The map was processed at super resolution pixel size
Voxel sizeX=Y=Z: 0.655 Å
Density
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.04
Minimum - Maximum-0.4288977 - 0.6876844
Average (Standard dev.)-9.447558e-05 (±0.019309342)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 251.51999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6550.6550.655
M x/y/z384384384
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/NS384384384
D min/max/mean-0.4290.688-0.000

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Supplemental data

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Additional map: EPYC1(49-72) peptide-bound Rubisco. The map was processed at...

Fileemd_22308_additional_1.map
AnnotationEPYC1(49-72) peptide-bound Rubisco. The map was processed at 2x binning of super resolution pixel size. The resolution reaches Nyquist frequency.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: EPYC1(49-72) peptide-bound Rubisco. The map was derived right...

Fileemd_22308_additional_2.map
AnnotationEPYC1(49-72) peptide-bound Rubisco. The map was derived right after 3DRefine at super resolution 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 : EPYC1(49-72) peptide-bound Rubisco

EntireName: EPYC1(49-72) peptide-bound Rubisco
Components
  • Complex: EPYC1(49-72) peptide-bound Rubisco
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small chain 2, chloroplastic
    • Protein or peptide: LCI5
  • Ligand: water

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Supramolecule #1: EPYC1(49-72) peptide-bound Rubisco

SupramoleculeName: EPYC1(49-72) peptide-bound Rubisco / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Chlamydomonas reinhardtii (plant)

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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

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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

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Macromolecule #3: LCI5

MacromoleculeName: LCI5 / type: protein_or_peptide / ID: 3 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 2.828148 KDa
SequenceString:
TNRVSPTRSV LPANWRQELE SLRN

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 1198 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 22500
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: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7462 pixel / Digitization - Frames/image: 1-50 / Number real images: 2500 / Average exposure time: 10.0 sec. / Average electron dose: 58.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1809869
CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 945755
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1gk8

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7jfo:
EPYC1(49-72)-bound Rubisco

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