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- EMDB-50109: cryo-EM structure of carboxysomal mini-shell icosahedral assembly... -

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Basic information

Entry
Database: EMDB / ID: EMD-50109
Titlecryo-EM structure of carboxysomal mini-shell icosahedral assembly from co-expression of CsoS1C, CsoS4A, and CsoS2-C (T = 9)
Map dataUnsharpened map
Sample
  • Complex: T=9 Minishell carboxysome complex of S4A and S1C shell subunits and CsoS2 scaffolding protein
    • Protein or peptide: Carboxysome shell vertex protein CsoS4A
    • Protein or peptide: Carboxysome shell protein CsoS1C
    • Protein or peptide: Carboxysome assembly protein CsoS2B
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsCarboxysome / mini-shell / Halothiobacillus neopolitanus / STRUCTURAL PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.8 Å
AuthorsNg PC / Li T / Basle A / Marles-Wright J / Liu LN
Funding support United Kingdom, 3 items
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, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50109.png

Downloads & links

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Map

FileDownload / File: emd_50109.map.gz / Format: CCP4 / Size: 2.7 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 896 pix.
= 742.784 Å		0.83 Å/pix.
x 896 pix.
= 742.784 Å		0.83 Å/pix.
x 896 pix.
= 742.784 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.829 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.049487 - 0.3057354
Average (Standard dev.)0.0003499746 (±0.011322945)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions896896896
Spacing896896896
CellA=B=C: 742.784 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50109_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map

Fileemd_50109_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_50109_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_50109_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : T=9 Minishell carboxysome complex of S4A and S1C shell subunits a...

EntireName: T=9 Minishell carboxysome complex of S4A and S1C shell subunits and CsoS2 scaffolding protein
Components
  • Complex: T=9 Minishell carboxysome complex of S4A and S1C shell subunits and CsoS2 scaffolding protein
    • Protein or peptide: Carboxysome shell vertex protein CsoS4A
    • Protein or peptide: Carboxysome shell protein CsoS1C
    • Protein or peptide: Carboxysome assembly protein CsoS2B
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: T=9 Minishell carboxysome complex of S4A and S1C shell subunits a...

SupramoleculeName: T=9 Minishell carboxysome complex of S4A and S1C shell subunits and CsoS2 scaffolding protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Halothiobacillus neapolitanus (bacteria)
Molecular weightTheoretical: 9.4 MDa

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Macromolecule #1: Carboxysome shell vertex protein CsoS4A

MacromoleculeName: Carboxysome shell vertex protein CsoS4A / type: protein_or_peptide / ID: 1 / Details: CsoS4A / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Halothiobacillus neapolitanus (bacteria)
Molecular weightTheoretical: 8.900287 KDa
Recombinant expressionOrganism: Escherichia coli BW25113 (bacteria)
SequenceString:
MKIMQVEKTL VSTNRIADMG HKPLLVVWEK PGAPRQVAVD AIGCIPGDWV LCVGSSAARE AAGSKSYPSD LTIIGIIDQW NGE

UniProtKB: Carboxysome shell vertex protein CsoS4A

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Macromolecule #2: Carboxysome shell protein CsoS1C

MacromoleculeName: Carboxysome shell protein CsoS1C / type: protein_or_peptide / ID: 2 / Details: Cso1A / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Halothiobacillus neapolitanus (bacteria)
Molecular weightTheoretical: 9.930453 KDa
Recombinant expressionOrganism: Escherichia coli BW25113 (bacteria)
SequenceString:
MAAVTGIALG MIETRGLVPA IEAADAMTKA AEVRLVGRQF VGGGYVTVLV RGETGAVNAA VRAGADACER VGDGLVAAHI IARVHSEVE NILPKAPEA

UniProtKB: Carboxysome shell protein CsoS1C

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Macromolecule #3: Carboxysome assembly protein CsoS2B

MacromoleculeName: Carboxysome assembly protein CsoS2B / type: protein_or_peptide / ID: 3 / Details: CsoS2-C / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Halothiobacillus neapolitanus (bacteria)
Molecular weightTheoretical: 29.358428 KDa
Recombinant expressionOrganism: Escherichia coli BW25113 (bacteria)
SequenceString: MPFCTSTPEP EAQSTEQSLT CEGQIISGTS VDASDLVTGN EIGEQQLISG DAYVGAQQTG CLPTSPRFNQ TGNVQSMGFK NTNQPEQNF APGEVMPTDF SIQTPARSAQ NRITGNDIAP SGRITGPGML ATGLITGTPE FRHAARELVG SPQPMAMAMA N RNKAAQAP ...String:
MPFCTSTPEP EAQSTEQSLT CEGQIISGTS VDASDLVTGN EIGEQQLISG DAYVGAQQTG CLPTSPRFNQ TGNVQSMGFK NTNQPEQNF APGEVMPTDF SIQTPARSAQ NRITGNDIAP SGRITGPGML ATGLITGTPE FRHAARELVG SPQPMAMAMA N RNKAAQAP VVQPEVVATQ EKPELVCAPR SDQMDRVSGE GKERCHITGD DWSVNKHITG TAGQWASGRN PSMRGNARVV ET SAFANRN VPKPEKPGSK ITGSSGNDTQ GSLITYSGGA RG

UniProtKB: Carboxysome assembly protein CsoS2B

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 157 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

Concentration0.85 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMTrisTris
1.0 mMEDTAEthylenediaminetetraacetic acid
10.0 mMMgCl2Magnesium chloride
20.0 mMNaHCO3Sodium bicarbonate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 70.0 K / Max: 70.0 K
Specialist opticsEnergy filter - Name: GIF 200 / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 19986 / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1229909 / Details: Blob based picking
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 131477
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Details: Ab initio model from cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Details: Homogeneous refinement job
Final 3D classificationNumber classes: 5 / Avg.num./class: 50000
Details: 3D classification using multiple ab initio models on curated dataset of 296626 particles. Icosahedral class taken forward for final refinement.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8b12


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial manual fitting of 8B12 into map followed by refinement using fit in map tool. Comprehensive real space refinement in Phenix real space refine.
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9f0h:
cryo-EM structure of carboxysomal mini-shell icosahedral assembly from co-expression of CsoS1C, CsoS4A, and CsoS2-C (T = 9)

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