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- PDB-6hbc: Structure of the repeat unit in the network formed by CcmM and Ru... -

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Basic information

Entry
Database: PDB / ID: 6hbc
TitleStructure of the repeat unit in the network formed by CcmM and Rubisco from Synechococcus elongatus
Components
  • Carbon dioxide concentrating mechanism protein CcmM
  • Ribulose 1,5-bisphosphate carboxylase small subunit
  • Ribulose bisphosphate carboxylase large chain
KeywordsPROTEIN BINDING / CcmM / M58 / M35 / SSUL domain / Rubisco / Carboxysome
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 1,5 Bisphosphate Carboxylase/Oxygenase / Ribulose bisphosphate carboxylase, small subunit / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain / RuBisCO large subunit, N-terminal domain / Ribulose bisphosphate carboxylase small subunit, domain / Ribulose bisphosphate carboxylase, small subunit superfamily / Ribulose bisphosphate carboxylase, small chain ...Carboxysome assembly protein CcmM / : / Ribulose bisphosphate carboxylase, small subunit / Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase / Ribulose bisphosphate carboxylase, small subunit / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain / RuBisCO large subunit, N-terminal domain / 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 / Alpha-Beta Plaits / TIM Barrel / Alpha-Beta Barrel / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Carboxysome assembly protein CcmM / Ribulose bisphosphate carboxylase small subunit / Ribulose bisphosphate carboxylase large chain
Similarity search - Component
Biological speciesSynechococcus elongatus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.78 Å
AuthorsWang, H. / Yan, X. / Aigner, H. / Bracher, A. / Nguyen, N.D. / Hee, W.Y. / Long, B.M. / Price, G.D. / Hartl, F.U. / 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, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 12, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 23, 2019Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_DOI
Revision 1.2Feb 6, 2019Group: Data collection / Database references / Category: citation
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Feb 20, 2019Group: Data collection / Database references
Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Carbon dioxide concentrating mechanism protein CcmM
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose 1,5-bisphosphate carboxylase small subunit
E: Ribulose 1,5-bisphosphate carboxylase small subunit


Theoretical massNumber of molelcules
Total (without water)142,2825
Polymers142,2825
Non-polymers00
Water0
1
A: Carbon dioxide concentrating mechanism protein CcmM
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose 1,5-bisphosphate carboxylase small subunit
E: Ribulose 1,5-bisphosphate carboxylase small subunit

A: Carbon dioxide concentrating mechanism protein CcmM
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose 1,5-bisphosphate carboxylase small subunit
E: Ribulose 1,5-bisphosphate carboxylase small subunit

A: Carbon dioxide concentrating mechanism protein CcmM
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose 1,5-bisphosphate carboxylase small subunit
E: Ribulose 1,5-bisphosphate carboxylase small subunit

A: Carbon dioxide concentrating mechanism protein CcmM
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose 1,5-bisphosphate carboxylase small subunit
E: Ribulose 1,5-bisphosphate carboxylase small subunit


Theoretical massNumber of molelcules
Total (without water)569,12920
Polymers569,12920
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation3

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Components

#1: Protein Carbon dioxide concentrating mechanism protein CcmM


Mass: 10550.686 Da / Num. of mol.: 1 / Fragment: SSUL domain 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (strain PCC 7942) (bacteria)
Gene: ccmM, Synpcc7942_1423 / Plasmid: pHue / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q03513
#2: Protein Ribulose bisphosphate carboxylase large chain / RuBisCO large subunit


Mass: 52516.605 Da / Num. of mol.: 2 / Fragment: Rubisco large subunit
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (strain PCC 7942) (bacteria)
Gene: cbbL, rbcL, Synpcc7942_1426 / Plasmid: pET11a / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q31NB3, ribulose-bisphosphate carboxylase
#3: Protein Ribulose 1,5-bisphosphate carboxylase small subunit


Mass: 13349.196 Da / Num. of mol.: 2 / Fragment: Rubisco small subunit
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (strain PCC 7942) (bacteria)
Gene: Synpcc7942_1427 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q31NB2, ribulose-bisphosphate carboxylase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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
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.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Synechococcus elongatus PCC 7942 (bacteria) / Organelle: Carboxysome
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: Solutions were made fresh
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMPotassium chlorideKCl1
250 mMTris(HOCH2)3CNH21
310 mMMagnesium acetateMg(CH3COO)21
45 mMDTTC4H10O2S21
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample contained 10 nm gold beads and was not monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K / Details: blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 0.15 sec. / Electron dose: 1.05 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7UCSF Chimeramodel fitting
9REFMAC5.8.0155model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78916 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Average Fourier shell correlation
Details: The 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 ...Details: The 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.

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