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PLANT PHYSIOLOGYBiochemistryPeer-reviewed journal article

Rubisco packaging and stoichiometric composition of a native β-carboxysome

Yaqi SuniD, Yuewen ShengiD, Tao NiiD, Xingwu GeiD, Joscelyn SarsbyiD, Philip BrownridgeiD, Kang LiiD, Nathan J. HardenbrookiD, Gregory F. DykesiD, Nichola Rockliffe, Claire E. EyersiD, Peijun ZhangiD, Lu‐Ning LiuiD

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Abstract

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO2-fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within {beta}-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the {beta}-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in {beta}-carboxysomes, which is instrumental for Rubisco encapsulation and {beta}-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire {beta}-carboxysome. This study and recent findings on the {beta}-carboxysome structure provide insights into the assembly principles and structural variation of {beta}-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (21 Sept 2024) · CC BY 4.0

Cryo-ET of 169 nm β-carboxysomes from low-light Synechococcus elongatus reveals ~639 Rubiscos in concentric layers with CcmM bound at RbcL grooves; QconCAT proteomics yields the complete absolute protein stoichiometry.

β-carboxysome diameter shifts with light and CO2 (144 to 208 nm), yet how Rubisco packing changes with size and the full protein composition of intact β-carboxysomes were unresolved, a barrier to building functional carboxysomes in heterologous hosts. Prior cryo-ET covered larger 193 nm particles and earlier abundance estimates used fluorescence tagging; this work combines cryo-ET with QconCAT absolute quantification on smaller, more uniform low-light particles.

Key results

  1. ShownInside isolated β-carboxysomes, Rubiscos form a paracrystalline array of four to nine concentric layers that follow the shell. Their 4-fold axes point mostly radially, and orientation in the outermost layer does not differ from inner layers. In situ cryo-ET of FIB-milled cells shows the same ring-like packing.

    Isolates fixed CO2 at 2.56 ± 0.16 μmol.mg^−1.min^−1 (n = 3); particles 169.0 ± 11.8 nm; 92.4% had 4-6 layers, five most frequent (44.3%); neighbour spacing 121.9 ± 31.4 Å vs 128-129 Å in α-carboxysomes; layers 1-6 hold about 1, 20, 71, 174, 308 and 407 Rubiscos. · Structure and assembly of Rubisco in isolated…; In situ cryoET of β-carboxysomes; CryoET sample preparation, data acquisition, and…

  2. ShownA 169 nm β-carboxysome holds about 639 Rubisco L8S8 complexes. That is more than α-carboxysomes from Cyanobium, H. neapolitanus and Prochlorococcus but fewer than 193 nm β-carboxysomes. Because Rubisco spacing stays about the same, the authors attribute the difference mainly to diameter.

    639 ± 194 Rubisco complexes per particle (n = 185); a layer model using sphere surface area (4πr^2) and ∼120 Å spacing, referenced to layer 4, matched observed counts for layers 2 and 3; authors estimate a 193 nm particle fits one more layer, over 400 extra Rubiscos and more than 1200 in total. · Structure and assembly of Rubisco in isolated…; Discussion; Figure legends

  3. ShownSubtomogram averaging of Rubisco inside native carboxysomes resolves an extra density in the RbcL dimer groove. It matches the small-subunit-like (SSUL) module of CcmM, giving direct evidence of CcmM binding inside the carboxysome. Only one SSUL fits per groove, in either an up or a down position.

    185 carboxysomes averaged with D4 symmetry; gold-standard 3.7 Å, final cisTEM map 3.5 Å; density on the 2-fold axis at half Rubisco intensity fits CcmM SSUL (PDB 6HBC) in upper or lower groove; four potential SSUL sites per Rubisco, with apparently low occupancy; deposited as PDB 9FWV / EMD-50836. · Structure and assembly of Rubisco in isolated…; CryoET sample preparation, data acquisition, and…; Data availability; Figure legends

  4. ShownQconCAT quantification gives the full composition of a ~529 MDa β-carboxysome. CcmK2 hexamers dominate the shell and CcmM35 far outnumbers CcmM58. There are only 6 CcmL pentamers, fewer than the 12 vertices of a canonical icosahedron and the 11 CsoS4 pentamers of α-carboxysomes.

    Copies per particle: CcmK2 hexamers 1975, CcmM35 730, Rubisco 639 (RbcL-based), CcmM58 112, CcaA hexamers 28, CcmK4 hexamers 21, CcmN 8, CcmP 5, RbcX dimers 3, CcmK3 hexamers 2, CcmO 1; Rubisco ∼68% and CcmK2/K3/K4 hexamers ∼25% of total MW. · Protein stoichiometry of β-carboxysomes; Discussion; Tables

  5. SuggestedThe shell-cargo linker CcmN is scarce and not present in a fixed ratio to CcmM58, unlike earlier in vitro estimates. The authors propose that CcmM's high ratio of short to long isoforms, compared with CsoS2 in α-carboxysomes, favours cargo packing and a large Rubisco core, so scaffold composition may set size.

    CcmN 8 copies (0.02% of MW) vs CcmM58 112 and CcmM35 730; CcmM35 and CcmM58 reported at 6% of total MW; CcmM short:long ratio nearly five times that of CsoS2 (Supplementary Table 4); shell-to-cargo ratios stay consistent between α- and β-carboxysomes despite size differences. · Protein stoichiometry of β-carboxysomes; Discussion

Methods, data and reproducibility

Intact β-carboxysomes purified from low-light Syn7942 were imaged by cryo-ET; Rubiscos were template-matched and subtomogram-averaged (emClarity) for layer, orientation and copy analysis, complemented by in situ cryo-ET of FIB-milled cells. Absolute protein copies came from QconCAT-standardized LC-MS normalized to cryo-ET Rubisco counts.

  • Sucrose-gradient-purified intact β-carboxysomes from Syn7942 cultures kept at 15 μE·m^−2·s^−1 illumination, analyzed by cryo-ET and subtomogram averaging · Synechococcus elongatus PCC 7942 · n = 185 β-carboxysomes (subtomogram averaging) · PDB 9FWV; EMDB EMD-50836
  • Cryo-FIB-milled lamellae of vitrified Syn7942 cells imaged by in situ cryo-ET · Synechococcus elongatus PCC 7942
  • Purified β-carboxysome isolates co-digested with isotope-labelled QconCAT standard for LC-MS absolute protein quantification · Synechococcus elongatus PCC 7942 · n = three biological replicates (Methods); four per Figure 4 legend
  • Purified β-carboxysomes assayed for 14CO2 fixation (Rubisco carboxylation activity) · Synechococcus elongatus PCC 7942 · n = n = 3 independently purified preparations
  • Negative-stain EM, SDS-PAGE and anti-RbcL/anti-CcmK2 immunoblots of isolated β-carboxysomes · Synechococcus elongatus PCC 7942 · n = SDS-PAGE from three biological replicates
  • Reference structures used as the template-matching map, the refined Rubisco model, and the CcmM SSUL fit · PDB 5NV3 (template); PDB 8BCM; PDB 6HBC (Rubisco-CcmM35)
Methods & tools
Cryo-electron tomography with subtomogram averaging, emClarity v1.5.0.4 / v1.6.2, IMOD v4.9.12, cisTEM (reconstruction within emClarity), Chimera (molmap template generation), SerialEM and Thermo Scientific Tomography 5, Cryo-FIB milling (Aquilos FIB/SEM), QconCAT standard (ALACAT/Qbrick design, wheat germ cell-free expression), Q Exactive HF LC-MS/MS, Proteome Discoverer v.2.4 with Mascot v.2.8, Skyline v23.1, 14CO2 fixation Rubisco activity assay
Software & environment
IMOD v4.9.12; emClarity v1.5.0.4 and v1.6.2 (cisTEM reconstruction); Chimera molmap; SerialEM; Tomography 5; Proteome Discoverer v.2.4; Mascot v.2.8; Skyline v23.1; Dionex Chromatography MS Link 2.14
Reproducibility gaps
Only the Rubisco STA map and model are deposited (PDB/EMDB). Raw tilt series, tomograms and LC-MS raw files have no stated repository; other data are said to be in the paper or supplement. The cet_toolbox script is described as an in-house tool.
Limitations (6)
  • Syn7942 β-carboxysomes are irregular in shape, so cryo-ET could not localize components other than Rubisco. Shell composition and the placement of minor cargo therefore rest on mass spectrometry and a schematic model. · stated by the authors
  • It remains unknown how low-abundance components such as CcmN and β-CA (CcaA) are incorporated, and what sets β-carboxysome size is unresolved. The scaffold-isoform-ratio explanation is only proposed. · stated by the authors
  • The authors caution that even their smaller low-light β-carboxysomes remain larger than previously reported α-carboxysomes, which complicates direct structural comparison between the two carboxysome lineages. · stated by the authors
  • The authors note that the architecture of mature carboxysomes alone cannot definitively separate α- from β-carboxysome assembly mechanisms. Their finding of no outer-layer orientation difference also conflicts with a recent β-carboxysome study; they attribute this to plasticity under different growth conditions. · stated by the authors
  • Per-carboxysome copy numbers are normalized to the cryo-ET mean Rubisco count (639 ± 194), so the broad variation between particles carries into every absolute stoichiometry. Only one low-light growth condition was profiled. · noted in this summary
  • Replicate counts are inconsistent (three biological replicates in the proteomics Methods, four in the Figure 4 legend), and a Table 1 footnote ties CcmK3/CcmK4 and CcmO/CcmP values to paralog sequence homology, which tempers confidence in low-copy components. · noted in this summary

Read this if you study carboxysome or bacterial microcompartment assembly, need absolute protein copy numbers to engineer β-carboxysomes, or use in situ cryo-ET to analyze Rubisco packing.

Citations
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top 4%

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