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- EMDB-0180: Structure of the repeat unit in the network formed by CcmM and Ru... -

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Basic information

Entry
Database: EMDB / ID: EMD-0180
TitleStructure of the repeat unit in the network formed by CcmM and Rubisco from Synechococcus elongatus
Map dataMap after post-processing
Sample
  • Complex: Repeat unit in the CcmM-Rubisco network consisting of a SSUL domain from CcmM and each two RbcL and two RbcS chains from Rubisco
    • Protein or peptide: Carbon dioxide concentrating mechanism protein CcmM
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose 1,5-bisphosphate carboxylase small subunit
Function / homology
Function and homology information


structural constituent of carboxysome shell / photorespiration / carboxysome / ribulose-bisphosphate carboxylase / carbon fixation / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / photosynthesis / monooxygenase activity / magnesium ion binding
Similarity search - Function
Carboxysome assembly protein CcmM / : / 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. ...Carboxysome assembly protein CcmM / : / 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 / Trimeric LpxA-like superfamily
Similarity search - Domain/homology
Carboxysome assembly protein CcmM / Ribulose bisphosphate carboxylase small subunit / Ribulose bisphosphate carboxylase large chain
Similarity search - Component
Biological speciesSynechococcus elongatus PCC 7942 (bacteria) / Synechococcus elongatus (strain PCC 7942) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.78 Å
AuthorsWang H / Yan X / Aigner H / Bracher A / Nguyen ND / Hee WY / Long BM / Price GD / Hartl FU / Hayer-Hartl M
CitationJournal: Nature / Year: 2019
Title: Rubisco condensate formation by CcmM in β-carboxysome biogenesis.
Authors: H Wang / X Yan / H Aigner / A Bracher / N D Nguyen / W Y Hee / B M Long / G D Price / F U Hartl / M Hayer-Hartl /
Abstract: Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency. The α- and β-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate ...Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency. The α- and β-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-a complex of eight large (RbcL) and eight small (RbcS) subunits-and carbonic anhydrase. As HCO can diffuse through the proteinaceous carboxysome shell but CO cannot, carbonic anhydrase generates high concentrations of CO for carbon fixation by Rubisco. The shell also prevents access to reducing agents, generating an oxidizing environment. The formation of β-carboxysomes involves the aggregation of Rubisco by the protein CcmM, which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain. It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS. Here we have reconstituted the Rubisco-CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid-liquid phase separation in eukaryotes. Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC1. Understanding carboxysome biogenesis will be important for efforts to engineer CO-concentrating mechanisms in plants.
History
DepositionAug 10, 2018-
Header (metadata) releaseSep 12, 2018-
Map releaseDec 12, 2018-
UpdateFeb 20, 2019-
Current statusFeb 20, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0725
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0725
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hbc
  • Surface level: 0.0725
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0180.png

Downloads & links

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Map

FileDownload / File: emd_0180.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap after post-processing
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.0725 / Movie #1: 0.0725
Minimum - Maximum-0.28270108 - 0.49959758
Average (Standard dev.)0.00016535513 (±0.013774122)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 210.432 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8220.8220.822
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z210.432210.432210.432
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2830.5000.000

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Supplemental data

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Sample components

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Entire : Repeat unit in the CcmM-Rubisco network consisting of a SSUL doma...

EntireName: Repeat unit in the CcmM-Rubisco network consisting of a SSUL domain from CcmM and each two RbcL and two RbcS chains from Rubisco
Components
  • Complex: Repeat unit in the CcmM-Rubisco network consisting of a SSUL domain from CcmM and each two RbcL and two RbcS chains from Rubisco
    • Protein or peptide: Carbon dioxide concentrating mechanism protein CcmM
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose 1,5-bisphosphate carboxylase small subunit

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Supramolecule #1: Repeat unit in the CcmM-Rubisco network consisting of a SSUL doma...

SupramoleculeName: Repeat unit in the CcmM-Rubisco network consisting of a SSUL domain from CcmM and each two RbcL and two RbcS chains from Rubisco
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: CcmM contains a succession of three highly similar SSUL domains (residues 225-313, 340-428 and 455-539, respectively), which bind to cleft on the surface of Rubisco. Rubisco is a hexadecamer ...Details: CcmM contains a succession of three highly similar SSUL domains (residues 225-313, 340-428 and 455-539, respectively), which bind to cleft on the surface of Rubisco. Rubisco is a hexadecamer of eight RbcL and eight RbcS subunits. The complex has D4 symmetry. The SSUL-RbcL2-RbcS2 repeat units can have one of two orientations (up or down). Thus Rubisco complexes saturated with SSUL domains can have four different configurations (uuuu, uuud, uudd, udud). In reality, some SSUL binding sites are probably left unoccupied. The network is formed by flexible linkers connecting the SSUL domains in CcmM, which then interlink Rubisco hexadecamers.
Source (natural)Organism: Synechococcus elongatus PCC 7942 (bacteria) / Organelle: Carboxysome
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Carbon dioxide concentrating mechanism protein CcmM

MacromoleculeName: Carbon dioxide concentrating mechanism protein CcmM / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechococcus elongatus (strain PCC 7942) (bacteria)
Molecular weightTheoretical: 10.550686 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SEFLSSEVIT QVRSLLNQGY RIGTEHADKR RFRTSSWQPC APIQSTNERQ VLSELENCLS EHEGEYVRLL GIDTNTRSRV FEALIQRPD GSV

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Macromolecule #2: Ribulose bisphosphate carboxylase large chain

MacromoleculeName: Ribulose bisphosphate carboxylase large chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Synechococcus elongatus (strain PCC 7942) (bacteria)
Molecular weightTheoretical: 52.516605 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPKTQSAAGY KAGVKDYKLT YYTPDYTPKD TDLLAAFRFS PQPGVPADEA GAAIAAESST GTWTTVWTDL LTDMDRYKGK CYHIEPVQG EENSYFAFIA YPLDLFEEGS VTNILTSIVG NVFGFKAIRS LRLEDIRFPV ALVKTFQGPP HGIQVERDLL N KYGRPMLG ...String:
MPKTQSAAGY KAGVKDYKLT YYTPDYTPKD TDLLAAFRFS PQPGVPADEA GAAIAAESST GTWTTVWTDL LTDMDRYKGK CYHIEPVQG EENSYFAFIA YPLDLFEEGS VTNILTSIVG NVFGFKAIRS LRLEDIRFPV ALVKTFQGPP HGIQVERDLL N KYGRPMLG CTIKPKLGLS AKNYGRAVYE CLRGGLDFTK DDENINSQPF QRWRDRFLFV ADAIHKSQAE TGEIKGHYLN VT APTCEEM MKRAEFAKEL GMPIIMHDFL TAGFTANTTL AKWCRDNGVL LHIHRAMHAV IDRQRNHGIH FRVLAKCLRL SGG DHLHSG TVVGKLEGDK ASTLGFVDLM REDHIEADRS RGVFFTQDWA SMPGVLPVAS GGIHVWHMPA LVEIFGDDSV LQFG GGTLG HPWGNAPGAT ANRVALEACV QARNEGRDLY REGGDILREA GKWSPELAAA LDLWKEIKFE FETMDKL

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Macromolecule #3: Ribulose 1,5-bisphosphate carboxylase small subunit

MacromoleculeName: Ribulose 1,5-bisphosphate carboxylase small subunit / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Synechococcus elongatus (strain PCC 7942) (bacteria)
Molecular weightTheoretical: 13.349196 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSMKTLPKER RFETFSYLPP LSDRQIAAQI EYMIEQGFHP LIEFNEHSNP EEFYWTMWKL PLFDCKSPQQ VLDEVRECRS EYGDCYIRV AGFDNIKQCQ TVSFIVHRPG RY

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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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMKClPotassium chloride
50.0 mM(HOCH2)3CNH2Tris
10.0 mMMg(CH3COO)2Magnesium acetate
5.0 mMC4H10O2S2DTT

Details: Solutions were made fresh
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 3 seconds before plunging.
DetailsThis sample contained 10 nm gold beads and was not monodisperse

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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
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average exposure time: 0.15 sec. / Average electron dose: 1.05 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.78 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78916
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe cryoEM density for the repeat unit was masked by Refmac to the coordinates and converted into structure factors by Refmac. The model was adjusted with coot. This model was submitted to restrained refinement with Refmac against the structure factors.
RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Target criteria: Average Fourier shell correlation
Output model

PDB-6hbc:
Structure of the repeat unit in the network formed by CcmM and Rubisco from Synechococcus elongatus

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