Benjamin Engel

The structure of the COPI coat determined within the cell

The structure of the COPI coat determined within the cell

This paper was featured as an Editors' Choice in Science: http://science.sciencemag.org/content/359/6372/173.1

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

Pyrenoid tubules form conduits between thylakoid stacks and the pyrenoid

Mackinder et al., Cell, 2017 (https://www.ncbi.nlm.nih.gov/pubmed/28938113 ) identified several new proteins that appear to specifically co-localize with the pyrenoid tubules. Some of these proteins may play a role in establishing the intricate architecture of the pyrenoid tubules and minitubules.

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

However, symmetry-free subtomogram averaging of 45.6 nm3 subvolumes revealed that the RuBisCO complexes were actually hexagonally packed (Figure 12B).

Freeman Rosenzweig et al., Cell, 2017 (https://www.ncbi.nlm.nih.gov/pubmed/28938114 ) discovered that the pyrenoid matrix is not a static crystalline lattice, but rather mixes like a liquid. Due to the limitations of the CCD camera used for cryo-ET, Engel et al., eLife, 2015 (this paper) only examined the average neighborhood organization of larger cellular volumes containing multiple Rubisco holoenzymes. In contrast, Freeman Rosenzweig et al., 2017 took advantage of the resolving-power of a direct detector camera to precisely localize each individual Rubisco holoenzyme within the pyrenoid. This higher-resolution cryo-ET study found that although the average organization of the pyrenoid matrix resembles hexagonal close packing (HCP), as reported in Engel et al., 2015, this average does not represent the true local neighborhood around each Rubisco. The true packing of pyrenoid Rubisco is much more heterogeneous; it shows only short-range order and is highly similar to the organization of molecules within a liquid.

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

RuBisCO complexes within the pyrenoid have ∼15 nm between their centers (Figure 12D)

This estimation of ~15 nm spacing is similar to the more precise measurement in Freeman Rosenzweig et al., Cell, 2017 (https://www.ncbi.nlm.nih.gov/pubmed/28938114 ), which found that each Rubisco has an average of 4 to 5 neighbors that are 13.9 ± 1.5 nm away.

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

Figure 14

This tomogram was used in two new reviews on the eyespot: Deisseroth and Hegemann, Science, 2017 (https://www.ncbi.nlm.nih.gov/pubmed/28912215 ); and Thompson et al., Microbiology Monographs, 2017 (https://link.springer.com/chapter/10.1007/978-3-319-66365-4_9 ).

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

This attraction is proposed to involve van der Waals and electrostatic interactions between membrane surfaces (Barber and Chow, 1979; Chow et al., 2005), perhaps mediated by light-harvesting complexes bound to photosystem II (PSII) (Daum et al., 2010).

Puthiyaveetil et al., Nature Plants, 2017 (https://www.ncbi.nlm.nih.gov/pubmed/28263304 ) found that the balance of physicochemical forces on thylakoid membranes (attractive van der Waals forces and repulsive electrostatic and hydrostructural forces) can fully explain the observed stacking of higher plant grana. Repulsive electrostatic forces change significantly depending on the phosphorylation state of thylakoid proteins such as LHCII.

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

Technical advances such as direct detection cameras continue to improve the resolution of cryo-tomograms. Once higher resolutions are attained, it will be possible to use template matching to determine whether RuBisCO complexes have a preferred orientation relative to each other

While the new direct detector-enabled analysis of Rubisco positions was published in Freeman Rosenzweig et al., Cell, 2017 (https://www.ncbi.nlm.nih.gov/pubmed/28938114 ), the analysis of Rubsico orientations relative to their closest neighbors did not return a very clear answer, and thus was omitted from this publication. Some preferred orientations were found between neighbors, and filtering by these orientations enabled the subtomogram averaging of "dimer pairs" of Rubisco holoenzymes that appeared to be linked by a density (perhaps EPYC1). However, these pairs represented only a small percentage of the total Rubisco population in the pyrenoid matrix, so it is difficult to conclude anything about the significance of this observation. The full orientation analysis was published in the PhD thesis of Luis Kuhn Cuellar, which can be accessed here: https://mediatum.ub.tum.de/1327586

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

This data suggests that RuBisCO complexes within the pyrenoid may not directly interact with each other and that other factors, such as a dense matrix of proteins and carbohydrates or small linker proteins between the complexes (Meyer and Griffiths, 2013), determine the RuBisCO spacing.

Mackinder et al., PNAS, 2016 (https://www.ncbi.nlm.nih.gov/pubmed/27166422 ) characterized a relatively unstructured repeat protein called EPYC1, which is localized to the pyrenoid, binds Rubisco, and is required for pyrenoid formation. It is proposed that each of the four repeats in EPYC1 can bind Rubisco, perhaps at hydrophobic residues on α-helices of the Rubisco small subunit (Meyer et al., PNAS, 2012; https://www.ncbi.nlm.nih.gov/pubmed/23112177 ), linking Rubisco particles within the pyrenoid. In Freeman Rosenzweig et al., Cell, 2017 (https://www.ncbi.nlm.nih.gov/pubmed/28938114 ), comparison of the measured liquid-like organization of pyrenoid Rubisco to simulated data using different random packing models (single independent Rubisco particles, particle pairs, and linked networks) showed that the organization of the pyrenoid matrix most closely resembles a linked network with a preferred distance between neighbors (the neighbor's center position was placed 5.5-7 nm from the outer surface of the reference particle). It is plausible that EPYC1 enforces this preferred inter-particle distance by linking neighbor Rubisco holoenzymes.

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography