Overview of intact spinach chloroplasts visualized by cryo-ET.

A) Tomographic slice through an intact spinach chloroplast. B) Corresponding 3D segmentation of the volume. Characteristic elements are labeled. Thylakoid membranes (green) are organized into stacks of appressed thylakoids (grana) and individual thylakoids connecting the grana (stromal lamellae). Note the intact chloroplast envelope (blue) and single thylakoid surrounded by two plastoglobules (orange). Stroma particles (grey) are a mix of different complexes but predominantly Rubisco. C) The same thylakoid network as in B, here shown from the opposite side. Note the inclined stromal lamellae spiraling around the granum on the left and a smaller plastoglobule sandwiched between the membranes. D) Close-up on the margin of a granum (from a different tomogram) showing the individual thylakoids transitioning into inclined stromal lamellae. In the first transition at the top of the image, note the hole in the thylakoid where the stromal lamella meets the granum. E) Box plots showing the number of thylakoids per granum (left), grana diameter measured in TEM overviews and in tomograms (middle), and the ratio of appressed to non-appressed membrane surfaces measured in selected tomograms. Box: 75 percentile, yellow lines: mean, whiskers: 95 percentile. F) Representative segmentation and classification of appressed (teal) and non-appressed (yellow) thylakoid membranes (quantification shown in E).

Fine details of plastoglobules interacting with thylakoids.

A) Tomographic slice showing two plastoglobules: dense, spherical droplets that have surfaces covered with protein densities. B) 3D render of the surfaces of three plastoglobules. Note the ordered, small densities. Purple arrows in A and B indicate the protein coat. C) Gallery of tomographic slices showing plastoglobules in close proximity of thylakoids and the chloroplast envelope (second panel in the gallery of plastoglobules). D-E) Zoom-in on a segmentation of thylakoids with visible fenestrations in stromal lamellae. These holes may be caused by interactions with adjacent plastoglobules. F) Two examples of plastoglobules potentially interacting with thylakoids. Each example shows three consecutive tomographic z-slices. Note the protein-membrane interactions in example 2, with an array of small protein densities (potentially coming from the plastoglobule coat) mediating the contact between compartments.

Top view of the interconnections between two grana.

A) Averaged image of 40 tomographic slices (56 nm total averaged volume) from a tomogram denoised with the DeepDeWedge neural network [37] (high contrast, missing wedge inpainting), showing top views of two neighboring grana. Note the irregular shape of the grana, with wavy edges (traced with white line). Three stromal lamellae connect the two grana stacks. PSII particles are visible in the stacks. B) Close up of a region from A, showing stromal lamellae bridging two grana. Blue arrowheads: PSII particles in grana membranes.

Thylakoid spacing and thickness measurements.

Left: a zoom into thylakoids at the granum edge, showing the level of details observed in defocus cryo- tomograms denoised with cryo-CARE. Notice how the thylakoid tips stick to the appressed membrane, making them non-symmetrical. Right: quantification of thylakoid morphometric parameters. Membrane (1, blue) is the mean thickness of the lipid bilayer; stromal gap (2, green) is the mean distance between stacked thylakoids; lumen (3, yellow) is the mean thylakoid lumen width; thylakoid (4, red) is mean thickness of the thylakoid including two membrane bilayers plus the enclosed lumen (measured separately from the individual measurements of those features). Errors: SEM. Note the larger SEM values for lumen and thylakoid. This reflects the lumen variability between rigid appressed thylakoids of the granum and more labile stromal lamellae.

Lateral heterogeneity of PSII and PSI between appressed and non-appressed membranes.

A) Segmentation of the thylakoid network showing two close grana. Three membrane pieces are highlighted: M1, an appressed membrane of the granum (teal) transitions into a stromal lamellae membrane (yellow) and then into an end membrane of the second granum (yellow); M12, an appressed membrane in the middle of the granum; M23, a membrane that spans two grana with a small unstacked stromal lamella in the middle (zoomed-in and rotated in green inset panel A’) and at the right end. The eye with the arrow indicates the viewing direction in membranograms. B) Slice through the corresponding tomographic volume. C-E) Membranograms showing both the luminal and stromal sides of the three membranes highlighted in A. Color strips indicate the membrane domain (blue: appressed, yellow: non-appressed). Stromal lamellae and end membrane are annotated above membranogram. All membranograms show densities 2 nm above the membrane surface (see F for reference). There are sharp boundaries between densities in the appressed and non-appressed regions. PSII densities are seen only on the luminal side of the appressed membranes, whereas PSI densities are seen only on the stromal side of non-appressed membranes. F) Top: Diagram showing side views of structures (blue: PSII, orange: cytb6f, green: PSI, pink: ATPsyn) in a thylakoid membrane (grey). Bottom: membrane surface views showing densities that protrude from each structure 2 nm into the thylakoid lumen or stroma. G) Cartoon representation of the stromal and luminal surfaces of the membrane in E, with particle footprints pasted in (colored as in F).

Distribution and concentration of PSII in grana membranes.

A) Top: membranogram view of an appressed membrane piece with characteristic densities of dimers of PSII and cytb6f. Bottom: same membrane with models of PSII and cytb6f complexes fitted into the EM densities. B) Nearest neighbor distance plot of all the particles from the appressed membranes. Blue: PSII, orange: cytb6f, grey: all particles. Note that “all” also includes unassigned particles of unknown identity. The dotted line labeled PSII-NNSpin indicates the mean (± SEM) center-to-center distance between neighboring PSII cores. Nearest neighbor distances are much more variable between two cytb6f than between two PSII complexes. Identification of cytb6f particles was possible only in the best resolved membranes; therefore, particle number and distribution estimation are not as reliable. C) Segmentation of a granum with all detected PSII particles pasted in. Note that we did not analyze PSII distribution at the very tips of thylakoids; this is why the tips look unoccupied. D) Plot showing PSII nearest neighbor center-to-center distances (light blue) and concentration (dark blue) as a function of the particle position from the edge of the granum, in 10 nm bins (diagrammed above the plot). Dot size represents number of counts in each bin. E) Plot of the lengths of grana thylakoids selected for the analysis in D. Thylakoids of significantly different lengths were selected to minimize any potential effect of the membrane size on the PSII distribution.

Occupancy of PSII and PSII-LHCII supercomplexes in grana membranes.

A) Top panel: cartoon representation of an analyzed membrane patch (M4, fourth membrane in a granum) with footprints of all PSII core complexes (C2) placed into their respective positions. Middle panels: the same membrane patch with footprints of C2S2 and C2S2M2 PSII-LHCII supercomplexes placed in. Bottom panel: red outlines highlight possible in-plane clashes between different types of supercomplexes within the same membrane (outlines carried down from panels above). B) Quantification of the membrane coverage, depending on the type of supercomplexes placed in (the area of S2 LHCII trimers in supercomplexes is also shown). Reproduced results from the C. reinhardtii study (Chlamy) shown for comparison [2]. Error bars: SD. C) Cumulative membrane coverage of PSII supercomplexes throughout the stack. Each line in the plot represents the percentage of area occupied by PSII or different PSII-LHCII supercomplexes when adding membranes across the stack (from the first stacked membrane until the end of the granum). Line: mean, shading: SEM. Membranes from 15 grana were used for the analysis. D) Quantification of the in-plane clashes between PSII cores or different supercomplexes. PSII-PSII clash: 0.0002% (within error range); these might be complexes in direct contact like the example in A. E) Top: cartoon representation of the fifth membrane (M5) from the same granum with all C2S2 supercomplexes in place. Bottom, overlay of M4 (blue) and M5 (magenta) showing the overlap (white) between PSII-LHCII in appressed membranes separated by a stromal gap. F) Quantification of the stromal overlap between PSII cores, LHCII S2 trimers, and different supercomplex variants. For all plots, data was generated from 3 chloroplast preparations, using the best resolved membranes (n=173).

Overlap of densities across stacked membranes: intact vs. broken chloroplasts.

A) Tomographic slice showing a typical granum inside an intact chloroplast. White box marks the stack of membranes visualized in B. B) Above: segmentation of the piece of a granum from A with four membranes highlighted (M19-M22). Odd numbered membranes in magenta, even numbered in green. Below: corresponding membranogram overlays of the adjacent membrane pairs. Coloring represents all EM densities protruding ∼2 nm from the luminal side of each membrane. The three examples show two luminal overlaps (top and bottom) and an overlap across a stromal gap (middle). White: overlap between EM densities. C) Tomographic slice showing a part of a broken chloroplast, containing individual membranes and potentially re-stacked thylakoids. White box marks the pieces of two membranes from adjacent thylakoids. D) Top: membranogram from a piece of one appressed membrane in C, showing densities protruding ∼2 nm from the membrane’s luminal surface. Note the arrays of PSII complexes and absence of any other type of particles. Bottom: membranograms from the pair of appressed membranes (green and magenta) overlayed with each other. White: overlap between EM densities.

Sample preparation, tomogram selection on the FIB-milled lamellae, and types of tomograms collected.

A) Simplified schematic of the chloroplast preparation protocol, which aimed to minimize the time between chloroplast isolation and freezing. B) An overview of a typical FIB-milled lamella containing chloroplasts as seen in the TEM microscope. Chloroplasts can break and sometimes re-close during the isolation and plunging procedures. We tried to identify intact, spheroidal chloroplast sections in the lamellae and focused tomogram collection in those regions. White arrows point the grana stacks, visible in the overviews. C-F) Examples of tomograms collected in different regions of an intact chloroplast (C-E) as well as in a region containing a broken chloroplast (F).

Ultrastructure of grana sides and transition to stromal lamellae.

A) Segmentation of thylakoid membranes from two grana in close proximity. Note the irregular stacks and shifted thylakoids. B) View of the margin region on one side. Grana-forming thylakoids are flat and straight, whereas stromal lamellae are more undulating. C) View of the other side. Note the convoluted shape of stromal lamellae connecting with two other lamellae spanning from the same granum. The top part of the membrane segmentation was set to high transparency to improve visibility. D) Serial perpendicular slices through the granum, showing appressed membranes transitioning into stromal lamellae. Membranes merge and form bridges between each other.

Protein densities on two domains of thylakoids.

A) A 3D rendering of a thylakoid granum and stromal lamellae with tomographic densities projected onto the membrane surfaces (membranograms). The first membrane of the granum (M1) is separated from the stack to show the PSII and cyt.b6f densities in the appressed region of M2 below. Note that all non-appressed membranes (including grana margins, end membranes, and stromal lamellae) are populated with similar densities. B) and C) Zoom-in views of stromal lamellae extending from the side of the granum. The eye icon shows the viewing direction in C. All panels show membranes at the same threshold value and the same color scale.

Thylakoid-chloroplast envelope contacts.

Rare instances of thylakoids and thylakoid-like membranes approaching and contacting the inner envelope membrane inside intact chloroplasts. A-C) Tomographic slices showing overviews of the contact sites (white arrowheads). D-F) Zoom-ins of the contacts from respective images. Dashed box in B corresponds to the enlarged view in E.

Top view of a granum.

Tomographic slices from a tomogram denoised with DeepDeWedge program [37] (high contrast, missing wedge inpainting) showing the chloroplast stroma 15 nm above the granum (A), 7 nm above the granum (B), and 3 nm above the top membrane (C), as well as the luminal space between the top and second membranes of the granum (D). Note the high number of globular particles (mostly Rubisco) in A. Arrowheads indicate complexes appearing in the slices (Yellow: ribosomes, purple: ATPsyn, Green: putative PSI, Blue: PSII). Note the similar size of Rubisco and the ATPsyn F1 subunit, which complicates assignment. The tomographic volume was rotated so the top membrane was approximately parallel to the xy-plane.