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- PDB-9f0h: cryo-EM structure of carboxysomal mini-shell icosahedral assembly... -

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Basic information

Entry
Database: PDB / ID: 9f0h
Titlecryo-EM structure of carboxysomal mini-shell icosahedral assembly from co-expression of CsoS1C, CsoS4A, and CsoS2-C (T = 9)
Components
  • Carboxysome assembly protein CsoS2B
  • Carboxysome shell protein CsoS1C
  • Carboxysome shell vertex protein CsoS4A
KeywordsSTRUCTURAL PROTEIN / Carboxysome / mini-shell / Halothiobacillus neopolitanus
Function / homology
Function and homology information


structural constituent of carboxysome shell / carboxysome / carbon fixation / viral translational frameshifting
Similarity search - Function
Carboxysome assembly protein / Carboxysome shell peptide mid-region / Carboxysome shell vertex protein CsoS4A / Ethanolamine utilization protein EutN/carboxysome shell vertex protein CcmL / EutN/Ccml superfamily / Ethanolamine utilisation protein EutN/carboxysome / Bacterial microcompartment vertex (BMV) domain profile. / Bacterial microcompartments protein, conserved site / Bacterial microcompartment (BMC) domain signature. / CcmK/CsoS1, bacterial microcompartment domain ...Carboxysome assembly protein / Carboxysome shell peptide mid-region / Carboxysome shell vertex protein CsoS4A / Ethanolamine utilization protein EutN/carboxysome shell vertex protein CcmL / EutN/Ccml superfamily / Ethanolamine utilisation protein EutN/carboxysome / Bacterial microcompartment vertex (BMV) domain profile. / Bacterial microcompartments protein, conserved site / Bacterial microcompartment (BMC) domain signature. / CcmK/CsoS1, bacterial microcompartment domain / : / Bacterial microcompartment (BMC) domain profile. / BMC domain / Bacterial microcompartment domain / BMC / CcmK-like superfamily
Similarity search - Domain/homology
Carboxysome assembly protein CsoS2B / Carboxysome shell vertex protein CsoS4A / Carboxysome shell protein CsoS1C
Similarity search - Component
Biological speciesHalothiobacillus neapolitanus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.8 Å
AuthorsWang, P. / Marles-Wright, J. / Liu, L.N.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V009729/1 United Kingdom
Leverhulme TrustRPG-2021-286 United Kingdom
Royal SocietyURFR180030 United Kingdom
CitationJournal: Sci Adv / Year: 2024
Title: Molecular principles of the assembly and construction of a carboxysome shell.
Authors: Peng Wang / Jianxun Li / Tianpei Li / Kang Li / Pei Cing Ng / Saimeng Wang / Vincent Chriscoli / Arnaud Basle / Jon Marles-Wright / Yu-Zhong Zhang / Lu-Ning Liu /
Abstract: Intracellular compartmentalization enhances biological reactions, crucial for cellular function and survival. An example is the carboxysome, a bacterial microcompartment for CO fixation. The ...Intracellular compartmentalization enhances biological reactions, crucial for cellular function and survival. An example is the carboxysome, a bacterial microcompartment for CO fixation. The carboxysome uses a polyhedral protein shell made of hexamers, pentamers, and trimers to encapsulate Rubisco, increasing CO levels near Rubisco to enhance carboxylation. Despite their role in the global carbon cycle, the molecular mechanisms behind carboxysome shell assembly remain unclear. Here, we present a structural characterization of α-carboxysome shells generated from recombinant systems, which contain all shell proteins and the scaffolding protein CsoS2. Atomic-resolution cryo-electron microscopy of the shell assemblies, with a maximal size of 54 nm, unveil diverse assembly interfaces between shell proteins, detailed interactions of CsoS2 with shell proteins to drive shell assembly, and the formation of heterohexamers and heteropentamers by different shell protein paralogs, facilitating the assembly of larger empty shells. Our findings provide mechanistic insights into the construction principles of α-carboxysome shells and the role of CsoS2 in governing α-carboxysome assembly and functionality.
History
DepositionApr 16, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
4: Carboxysome shell vertex protein CsoS4A
A: Carboxysome shell protein CsoS1C
B: Carboxysome shell protein CsoS1C
C: Carboxysome shell protein CsoS1C
D: Carboxysome shell protein CsoS1C
E: Carboxysome shell protein CsoS1C
F: Carboxysome shell protein CsoS1C
H: Carboxysome shell protein CsoS1C
J: Carboxysome shell protein CsoS1C
X: Carboxysome assembly protein CsoS2B
Z: Carboxysome assembly protein CsoS2B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,08512
Polymers147,06111
Non-polymers241
Water2,828157
1
4: Carboxysome shell vertex protein CsoS4A
A: Carboxysome shell protein CsoS1C
B: Carboxysome shell protein CsoS1C
C: Carboxysome shell protein CsoS1C
D: Carboxysome shell protein CsoS1C
E: Carboxysome shell protein CsoS1C
F: Carboxysome shell protein CsoS1C
H: Carboxysome shell protein CsoS1C
J: Carboxysome shell protein CsoS1C
X: Carboxysome assembly protein CsoS2B
Z: Carboxysome assembly protein CsoS2B
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)8,825,104720
Polymers8,823,646660
Non-polymers1,45860
Water11,890660
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59

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Components

#1: Protein Carboxysome shell vertex protein CsoS4A


Mass: 8900.287 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CsoS4A
Source: (gene. exp.) Halothiobacillus neapolitanus (bacteria)
Gene: csoS4A, orfA, Hneap_0918 / Production host: Escherichia coli BW25113 (bacteria) / References: UniProt: O85043
#2: Protein
Carboxysome shell protein CsoS1C


Mass: 9930.453 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: Cso1A
Source: (gene. exp.) Halothiobacillus neapolitanus (bacteria)
Gene: csoS1C, Orf1, Hneap_0916 / Production host: Escherichia coli BW25113 (bacteria) / References: UniProt: P45688
#3: Protein Carboxysome assembly protein CsoS2B / Carboxysome shell protein CsoS2B


Mass: 29358.428 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: CsoS2-C
Source: (gene. exp.) Halothiobacillus neapolitanus (bacteria)
Gene: csoS2, Hneap_0920 / Production host: Escherichia coli BW25113 (bacteria) / References: UniProt: O85041
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 157 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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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: T=9 Minishell carboxysome complex of S4A and S1C shell subunits and CsoS2 scaffolding protein
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 9.4 MDa / Experimental value: NO
Source (natural)Organism: Halothiobacillus neapolitanus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTrisTris1
21 mMEthylenediaminetetraacetic acidEDTA1
310 mMMagnesium chlorideMgCl21
420 mMSodium bicarbonateNaHCO31
SpecimenConc.: 0.85 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingElectron dose: 46 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 19986
EM imaging opticsEnergyfilter name: GIF 200 / Energyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionDetails: Initial patch CTF correction for micrographs with per-group CTF corrections following 3D reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1229909 / Details: Blob based picking
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 1.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131477 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Initial manual fitting of 8B12 into map followed by refinement using fit in map tool. Comprehensive real space refinement in Phenix real space refine.
Atomic model buildingPDB-ID: 8B12

8b12


Accession code: 8B12 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 48.76 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00686762
ELECTRON MICROSCOPYf_angle_d0.57229161
ELECTRON MICROSCOPYf_chiral_restr0.04541100
ELECTRON MICROSCOPYf_plane_restr0.00441207
ELECTRON MICROSCOPYf_dihedral_angle_d5.1416999

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