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- EMDB-25228: H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25228
TitleH. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex
Map dataupsampled, density-modified map
Sample
  • Complex: H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex
    • Complex: Ribulose bisphosphate carboxylase large chain (E.C.4.1.1.39), Ribulose bisphosphate carboxylase small chain (E.C.4.1.1.39)
      • Protein or peptide: Ribulose bisphosphate carboxylase large chain
      • Protein or peptide: Ribulose bisphosphate carboxylase small chain
    • Complex: Carboxysome shell carbonic anhydrase (E.C.4.2.1.1)
      • Protein or peptide: Carboxysome shell carbonic anhydrase
  • Ligand: water
Keywordsrubisco / lyase / TIM-barrel / complex
Function / homology
Function and homology information


carboxysome / ribulose-bisphosphate carboxylase / carbon fixation / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / monooxygenase activity / carbonic anhydrase / carbonate dehydratase activity / magnesium ion binding / metal ion binding
Similarity search - Function
: / : / : / Carboxysome Shell Carbonic Anhydrase, catalytic domain / Carboxysome Shell Carbonic Anhydrase, N-terminal / Carboxysome shell carbonic anhydrase / Carboxysome shell carbonic anhydrase, N-terminal / Carboxysome shell carbonic anhydrase, C-terminal / Carboxysome Shell Carbonic Anhydrase, C-terminal / Ribulose bisphosphate carboxylase, small subunit ...: / : / : / Carboxysome Shell Carbonic Anhydrase, catalytic domain / Carboxysome Shell Carbonic Anhydrase, N-terminal / Carboxysome shell carbonic anhydrase / Carboxysome shell carbonic anhydrase, N-terminal / Carboxysome shell carbonic anhydrase, C-terminal / Carboxysome Shell Carbonic Anhydrase, C-terminal / 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 / Carboxysome shell carbonic anhydrase / Ribulose bisphosphate carboxylase small subunit
Similarity search - Component
Biological speciesHalothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.07 Å
AuthorsBlikstad C / Dugan E
Funding support United States, 1 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-SC00016240 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Identification of a carbonic anhydrase-Rubisco complex within the alpha-carboxysome.
Authors: Cecilia Blikstad / Eli J Dugan / Thomas G Laughlin / Julia B Turnšek / Mira D Liu / Sophie R Shoemaker / Nikoleta Vogiatzi / Jonathan P Remis / David F Savage /
Abstract: Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO concentrating mechanism (CCM). In the ...Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO by carbonic anhydrase, producing a high CO concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from . We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCA's binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.
History
DepositionOct 28, 2021-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25228.png

Downloads & links

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Map

FileDownload / File: emd_25228.map.gz / Format: CCP4 / Size: 54.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationupsampled, density-modified map
Voxel sizeX=Y=Z: 0.60059 Å
Density
Contour LevelBy AUTHOR: 0.37
Minimum - Maximum-0.93538415 - 2.109387
Average (Standard dev.)0.0007601123 (±0.17402714)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions242242242
Spacing242242242
CellA=B=C: 145.34273 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25228_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unmodified full map

Fileemd_25228_additional_1.map
Annotationunmodified full map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Carboxysomal rubisco/CsoSCA-peptide (1-50)complex

Fileemd_25228_half_map_1.map
AnnotationCarboxysomal rubisco/CsoSCA-peptide (1-50)complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Carboxysomal rubisco/CsoSCA-peptide (1-50)complex

Fileemd_25228_half_map_2.map
AnnotationCarboxysomal rubisco/CsoSCA-peptide (1-50)complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex

EntireName: H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex
Components
  • Complex: H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex
    • Complex: Ribulose bisphosphate carboxylase large chain (E.C.4.1.1.39), Ribulose bisphosphate carboxylase small chain (E.C.4.1.1.39)
      • Protein or peptide: Ribulose bisphosphate carboxylase large chain
      • Protein or peptide: Ribulose bisphosphate carboxylase small chain
    • Complex: Carboxysome shell carbonic anhydrase (E.C.4.2.1.1)
      • Protein or peptide: Carboxysome shell carbonic anhydrase
  • Ligand: water

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Supramolecule #1: H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex

SupramoleculeName: H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 533 KDa

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Supramolecule #2: Ribulose bisphosphate carboxylase large chain (E.C.4.1.1.39), Rib...

SupramoleculeName: Ribulose bisphosphate carboxylase large chain (E.C.4.1.1.39), Ribulose bisphosphate carboxylase small chain (E.C.4.1.1.39)
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Halothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)

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Supramolecule #3: Carboxysome shell carbonic anhydrase (E.C.4.2.1.1)

SupramoleculeName: Carboxysome shell carbonic anhydrase (E.C.4.2.1.1) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3

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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: 1 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Halothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)
Strain: ATCC 23641 / c2
Molecular weightTheoretical: 53.831723 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSAVKKYSAG VKEYRQTYWM PEYTPLDSDI LACFKITPQP GVDREEAAAA VAAESSTGTW TTVWTDLLTD MDYYKGRAYR IEDVPGDDA AFYAFIAYPI DLFEEGSVVN VFTSLVGNVF GFKAVRGLRL EDVRFPLAYV KTCGGPPHGI QVERDKMNKY G RPLLGCTI ...String:
MSAVKKYSAG VKEYRQTYWM PEYTPLDSDI LACFKITPQP GVDREEAAAA VAAESSTGTW TTVWTDLLTD MDYYKGRAYR IEDVPGDDA AFYAFIAYPI DLFEEGSVVN VFTSLVGNVF GFKAVRGLRL EDVRFPLAYV KTCGGPPHGI QVERDKMNKY G RPLLGCTI KPKLGLSAKN YGRAVYECLR GGLDFTKDDE NINSQPFMRW RDRFLFVQDA TETAEAQTGE RKGHYLNVTA PT PEEMYKR AEFAKEIGAP IIMHDYITGG FTANTGLAKW CQDNGVLLHI HRAMHAVIDR NPNHGIHFRV LTKILRLSGG DHL HTGTVV GKLEGDRAST LGWIDLLRES FIPEDRSRGI FFDQDWGSMP GVFAVASGGI HVWHMPALVN IFGDDSVLQF GGGT LGHPW GNAAGAAANR VALEACVEAR NQGRDIEKEG KEILTAAAQH SPELKIAMET WKEIKFEFDT VDKLDTQNRW SHPQF EK

UniProtKB: Ribulose bisphosphate carboxylase large chain

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Macromolecule #2: Ribulose bisphosphate carboxylase small chain

MacromoleculeName: Ribulose bisphosphate carboxylase small chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Halothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)
Strain: ATCC 23641 / c2
Molecular weightTheoretical: 12.866575 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAEMQDYKQS LKYETFSYLP PMNAERIRAQ IKYAIAQGWS PGIEHVEVKN SMNQYWYMWK LPFFGEQNVD NVLAEIEACR SAYPTHQVK LVAYDNYAQS LGLAFVVYRG N

UniProtKB: Ribulose bisphosphate carboxylase small subunit

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Macromolecule #3: Carboxysome shell carbonic anhydrase

MacromoleculeName: Carboxysome shell carbonic anhydrase / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: carbonic anhydrase
Source (natural)Organism: Halothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)
Molecular weightTheoretical: 5.690505 KDa
SequenceString:
MNTRNTRSKQ RAPFGVSSSV KPRLDLIEQA PNPAYDRHPA CITLPERTCR

UniProtKB: Carboxysome shell carbonic anhydrase

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 200 / 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

Concentration0.2665 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC4H11NO3Tris
80.0 mMNaClSodium chloridesodium chloride
2.0 %C3H8O3glycerol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 3 seconds.
Details0.5 uM of rubisco and 0.5 mM of peptide

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 57000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5742 / Average electron dose: 50.0 e/Å2 / Details: 3 x 3 multishot image shift pattern.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Stochastic gradient descent
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.07 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 52375
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Map-model FSC and geometry
Output model

PDB-7snv:
H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex

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