EMBO/soda-nel huggingface dataset:
LIN28A loss-of-function is associated with Parkinson's disease pathogenesis (2019)
Figure 2

Fig. 2. Human embryonic stem cells (hESCs) carrying the LIN28A(R192G) mutation exhibit defects in self-renewal, shape, and phenotype. (A, B) Cell growth and population doubling levels (PDLs) during passaging. In (B), dots represent individual PDLs, boxes and bars represent mean ± SE of five PDL values of +/+ and +/m lines. *Significantly different from control with p<0.05, ANOVA. (C) Phase-contrast (left), alkaline phosphatase-stained (middle), and immunocytochemical images stained with pluripotent cell markers (right). Images were taken at cell passage P8 (+/+, +/m) or P6 (m/m). Scale bars, 100 µm. (D) Enriched-GO and KEGG pathways of differentially expressed genes (DEGs) in the mutant cells (m/m c38 and +/m c43) vs +/+ (C42) (extended data in Appendix Figure. S3). (E) Unsupervised hierarchical clustering heatmap of the gene expression (ratios of mutant/WT) categorized in "neurodegenerative disorders" ('Parkinson's disease', 'Alzheimer's disease', and 'Huntington's disease').
LIN28A loss-of-function is associated with Parkinson's disease pathogenesis (2019)
Figure 3

Fig. 3. Neural induction of wild-type (WT) and mutant (LIN28A(R192G)) human embryonic stem cells (hESCs). (A) Neural differentiation of hESCs was induced on a feeder layer of MS5 stromal cells. Representative colonies differentiated from the WT (+/+, c42) and homozygous mutant (m/m, c38) hESCs are shown (left). Neural induction efficiencies were estimated by % neuroepithelial colonies out of total colonies (n=5~7 dishes for each group). Neuroepithelial cell colonies were identified by neuroepithelial cell structure (neural rosettes) and by staining of markers specific for primitive neuroepithelial cells (PLZF, N-CADHERIN, and SOX2) and rostral embryonic brain (OTX2). Scale bars, 100 µm. (B) Ventral midbrain (VM) patterning was assessed during 24 days of +/+ and +/m hESC differentiation (chemically defined protocol). Each dot represents mRNA expression detected by qPCR; boxes and bars represent means and SEs of +/+ and +/m lines. The gene expression levels of +/+ and +/m at day 24 are compared in the last graph (n=4, *p<0.05 or **p<0.001, t-test). (C) Neural stem cell (NSC) expansion estimated by cell growth curve and PDL during NSC passages (n=12, *p<0.001, t-test). NSCs were derived from neuroepithelial cell colonies (shown in (A)).
LIN28A loss-of-function is associated with Parkinson's disease pathogenesis (2019)
Figure 4

Fig. 4. Parkinson's disease (PD) manifestations in terminally differentiated neuron cultures. The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. (A) General cell survival (calcein-AM)/death (EthD-1). (B) Cleaved caspase-3+ apoptotic cell death. (C) Cellular senescence (β-gal staining). (D) DNA damage estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. (E) Mitochondrial ROS (MitoSox). (F) Mitochondrial membrane potential (MitoID). (G) Seahorse test. Terminal differentiation of neural stem cells (NSCs) was induced and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed on the NSCs at differentiation day 4. p<0.001*, n=7 (WT), 10 (mutant), t-test. (H) Western blot analyses for ER stress marker proteins. (I) Resistance of mDA neurons against mitochondrial toxins. Differentiated mDA neurons were exposed to the toxins indicated for 1 h, and viable TH+ mDA neurons were counted. Data are expressed as % TH+ cells relative to untreated. Three cultures were analyzed for each cell line. N=3 independent experiments, *Significance at p<0.05. Scale bars, 100 µm.
LIN28A loss-of-function is associated with Parkinson's disease pathogenesis (2019)
Figure 5

Fig. 5. Developmental defects and Parkinson's disease (PD) manifestation in midbrain-type dopamine (mDA) neurons derived from patient (pt)-human induced pluripotent stem cells (hiPSCs) are rescued by correction of the LIN28A mutation in the pt-hiPSCs. (A-C) Representative in vitro phase-contrast (A), TH/DAPI or TH (insets) (B), and TH/SYNAPSIN-stained (C) images in the cultures terminally differentiated from mutant (c13, c19) and corrected hiPSCs (c22, c59). (D-G) DA neuron differentiation, morphological, synaptical, and functional maturation of DA neurons were assessed by DA neuronal yields (D), TH+ fiber lengths (E), synaptic puncta densities (F), and presynaptic DA neuron releases (G), respectively. DA levels were measured in the media conditioned in the differentiated cultures for 24 h (D15) and evoked by KCl-mediated depolarization for 30 min. (H) DNA breaks estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. (I) SA-β-gal staining was performed for cellular senescence analysis. (J) Mitochondrial ROS (MitoSox) and (K) mitochondrial membrane potential (MitoID) were analyzed after terminal differentiation. Representative images for (H)~(K) are shown in Appendix Figure. S10. Significance at *p<0.05 or **p<0.001. (L) Accumulation of α-synuclein oligomers analyzed by western blotting. The mutant (c13) and corrected (c22) neural stem cells (NSCs) were transduced with α-synuclein-expressing lentiviruses. At 6 days after differentiation, cells were extracted and fractionated to Triton X-100 (1%)-soluble and insoluble fractions. Shown is the representative WB image of three independent experiments. α-Synuclein oligomers are indicated with a bracket, while monomer bands are marked with an arrowhead. (M) ClueGO/cytoscape for GO biological process upregulated in NSCs differentiated from the corrected iPSCs, c22 (vs the mutant pt-hiPSCs, c19). Term enrichment significance is represented by node size (Kappa score > 0.4). Most significant GO-terms are numbered and listed below. Also, see related Appendix Figure. S11. Scale bars, 50 μm (A, B), 20 μm (C).
LIN28A loss-of-function is associated with Parkinson's disease pathogenesis (2019)
Figure 6

Fig. 6. Behavioral and histologic assays of Parkinson's disease (PD) rats grafted with differentiated cells from mutant and corrected patient (pt)-human induced pluripotent stem cells (hiPSCs). (A) Amphetamine-induced rotation scores. The ipsilateral net rotation values of individual animals transplanted with differentiated mutant (n=7) and corrected (n=6) pt-hiPSCs are depicted. Significant differences from the mutant cell-transplanted at each post-transplantation time point are at *p<0.05 or **p<0.001. (B) Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05. (C) Overviews of hNCAM+/TH+ cell grafts at 8 weeks post-transplantation. Insets, TH+ DA neuron morphology in grafts shown with high magnification. (D) Co-expression of the mDA neuron-specific FOXA2, LMX1A, NURR1, DAT in TH+ DA cells generated by the corrected pt-hiPSC transplantation. (E) Total number of TH+ cells in the graft (TH+ numbers/mm3) per animal. Significance at **p<0.001, t-test. Scale bars, 500 μm (C), 20 μm (D).
LIN28A loss-of-function is associated with Parkinson's disease pathogenesis (2019)
Figure 7

Fig. 7. Loss-of-function mechanisms of LIN28A(R192G) mutation. (A) LIN28A protein stability assays. HEK293 cells were transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G. Protein samples were prepared during 24 h of cycloheximide (50 μg/ml) treatment. Representative western blot data for LIN28A protein level changes normalized to β-actin protein levels (upper) or with normalization based on initial (0 h) LIN28A protein levels (lower). Band intensities were quantified using ImageJ, and normalized values are depicted (lower graph) (n=5). (B) The proteasome inhibitor MG132 treatment increases LIN28A(R192G) protein level. After serum starvation for 3 hours, HEK293T cells expressing LIN28A(R912G) were treated with or without MG132 (0.1-10 uM) for 30 minutes, and then followed by WB analyses. (C) LC-MS/MS analysis. Immunoprecipitated FLAG-LIN28A WT was loaded on an SDS gel (left upper). Bold red sequences indicate peptides matching LIN28A (left lower). Right, LC-MS/MS spectra of WT LIN28A. (D) Cytoplasmic localization of WT LIN28A and LIN28A(R192G) proteins. HeLa cells transfected with pFLAG-WT-LIN28 and pFLAG-LIN28A(R192G) were stained with FLAG antibody and counterstained with DAPI. (E) LET-7 microRNA regulatory activity of LIN28A. HeLa cells were transfected with plasmids expressing FLAG-tagged WT- LIN28A, LIN28AR192G, or mock pFLAG vector (control), and Let-7a, b, and c levels were quantified by RT-qPCR. The level of RNA was normalized to that of U6 snRNA. p<0.05, n=26 from 6 independent experiments, ns = not significant, ANOVA. (F) Affinity of LIN28A protein binding to its cognate mRNA 3′UTR region. Upper: schematic for pmirGLO-LIN28A used in this assay, a bicistronic Firefly/Renilla luciferase (FLuc/RLuc) plasmid containing the part of the LIN28A 3′UTR containing the putative LIN28A protein binding sites (black dots)Wilbert et al., 2012(). Lower: HeLa cells were transiently co-transfected with pmirGLO-LIN28A 3′UTR along with pFLAG-WT-LIN28A, pFLAG-LIN28A R192G, or mock pFLAG control. LIN28A protein-mRNA binding activities were determined by relative luciferase activity levels of FLuc normalized to those of RLuc. *p<0.05, n=18 from 6 independent experiments. (G) LIN28A-RHA co-IP assays in HEK293 cells transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G.
ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria association (2016)
Figure 1

Figure 1. Expression of wild type and ALS/FTD mutant FUS reduces ER-mitochondria associations in NSC34 cells.

(A) expression of FUS does not alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in transfected NSC34 cells. Immunoblots of NSC34 cells transfected with EGFP as a control (CTRL), or wild-type or mutant EGFP-FUS. Transfected cells were purified via EGFP using a cell sorter and the samples probed on immunoblots as indicated. On the FUS immunoblot, samples were probed with FUS antibody to show endogenous and transfected proteins; tubulin is shown as a loading control. (B) representative electron micrographs of ER–mitochondria associations in NSC34 cells transfected with control EGFP vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K as indicated; arrowheads with loops show regions of association. Scale bar=200 nm. Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. N=27-30 cells and 247-424 mitochondria, error bars are s.e.m.; ***p<0.001. (C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (C) immunoblots of cells either mock transfected or treated with control (CTRL) or FUS siRNAs; GAPDH is shown as a loading control. (D) representative electron micrographs of ER-mitochondria associations in control (CTRL) and FUS siRNA treated cells. Arrowheads with loops show regions of association. Scale bar=200 nm. Data analysed by unpaired t-test. N=27-28 cells and 193-202 mitochondria.
ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria association (2016)
Figure 2

Figure 2. Expression of wild type and mutant FUS reduces ER-mitochondria associations and the VAPB-PTPIP51 interaction in NSC34 cells.

(A) FU-induced reductions in ER-mitochondria associations can be detected using SIM. NSC34 cells were transfected with either EGFP control vector, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and immunostained for TOM20 and PDI to label mitochondria (Mito) and ER respectively; FUS were detected via their EGFP tags. Merge (ZOOM) show zoomed images of boxed regions and co-localisation shows co-localised pixels. Scale bar=2 m. Bar chart shows ER-mitochondria co-localisation (Manders coefficient) normalized to control in the different samples. Data were analysed by one-way ANOVA with Tukey’s post-hoc test. 10-14 cells were analysed per condition from 3 independent experiments; error bars are s.e.m., **p<0.01 and ***p<0.001. (B) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted by wild-type and ALS/FTD mutant FUS. NSC34 cells were transfected with EGFP control vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and proximity ligation assays performed using VAPB and PTPIP51 antibodies. FUS were detected via their EGFP tags. Scale bar=10 m. Bar chart shows relative number of proximity ligation assay signals/cell. Data were analysed by one-way ANOVA and Tukey’s post-hoc test; n=47-53 cells from 5 experiments. Error bars are s.e.m.; ***p<0.001. (C) overexpression of FUS reduces the binding of VAPB to PTPIP51 in transfected cells. Cells were transfected as indicated with either control empty vector (CTRL), HA-PTPIP51+CTRL, or HA-PTPIP51+ either EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K. PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown and no immunoprecipitating VAPB signals were obtained in the absence of HA-PTPIP51. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals. Data were analysed by one-way ANOVA and Tukey’s post-hoc test; N=4. Error bars are s.e.m; *** p<0.001.
ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria association (2016)
Figure 3

Figure 3. Overexpression of FUS reduces ER-mitochondria associations and the VAPB-PTPIP51 interaction in spinal cords of FUS transgenic mice.

(A) overexpression of FUS does not affect expression of VAPB, PTPIP51 or mitofusin-2. Immunoblots of spinal cord proteins from three 10 week old FUS transgenic mice and three age-matched littermates are shown; FUS was detected via its HA-tag. Tubulin was used as a loading control. (B) representative electron micrographs of ER-mitochondria associations in lumbar spinal cord motor neurons of FUS transgenic mice and their non-transgenic littermates; arrowheads with loops show regions of association. Scale bar is 200 nm. Bar chart shows % of mitochondrial surface closely apposed to ER in the two samples. Data were analysed by unpaired t-test. N=67-88 cells and 438-749 mitochondria, error bars are s.e.m.; ***p<0.001. (C) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice. Representative images of proximity ligation signals in 11 week old FUS and non-transgenic (NTg) littermate mice. Data were analysed by unpaired t-test; N=40 cells from 3 FUS and 3 non-transgenic littermates (age 10 weeks). Error bars are s.e.m.; ***p<0.001.
ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria association (2016)
Figure 4

Figure 4. Expression of FUS disrupts cellular Ca2+ homeostasis and mitochondrial ATP production.

(A and B) FUS disrupts Ca2+ homeostasis. HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K as indicated. Release of ER Ca2+ was induced by treatment of cells with OxoM. (A) shows cytosolic Ca2+ levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right. Fluo4 fluorescence shows a transient increase in cytosolic Ca2+ levels upon OxoM treatment but compared to control, wild-type and mutant FUS all increase peak cytosolic Ca2+ levels. (B) shows mitochondrial Ca2+ levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right. Data were analysed by one-way ANOVA and Tukey’s post hoc test. (A) N=49-52 cells from 3 experiments; (B) N=50-52 cells from 5 experiments, error bars are s.e.m.; *p<0.05, **p<0.01, ***p<0.001. (C) FUS reduces mitochondrial ATP production. ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1.03 and either control vector (CTRL), HA-FUS, HA-FUSR521C or HA-FUSR518K. Cells were imaged in time-lapse prior to and after KCN treatment to inhibit oxidative phosphorylation. Representative traces of YFP/CFP ratios are shown for the different samples; initial YFP/CFP ratios prior to KCN treatment and those after KCN treatment are indicated. The fall in YFP/CFP ratios correlates with ATP produced by oxidative phosphorylation. Bar chart shows relative ATP levels produced by oxidative phosphorylation (OXPHOS) in the different samples. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N=29-54 cells from 5 experiments, error bars are s.e.m; *p<0.05, ***p<0.001.
ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria association (2016)
Figure 5

Figure 5. FUS activates GSK-3 in transfected cells and transgenic mice.

(A) cells were transfected with either control vector (CTRL), HA-FUS, HA-FUSR521C or HA FUSR518K and the samples probed on immunoblots for GSK-3 phosphorylated on serine-9 (GSK-3-S9), total GSK-3, FUS (using FUS antibody) and tubulin as a loading control. Phosphorylation of GSK-3 serine-9 is the principal mechanism for regulating its activity; serine-9 phosphorylation inhibits GSK-3 activity. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N=4, error bars are s.e.m; *p<0.05. (B) immunoblots of total and serine-9 phosphorylated GSK-3 in spinal cords from three 10 week-old FUS transgenic mice and their non-transgenic littermates. Samples were probed for tubulin as a loading control. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by unpaired t-test, error bars are s.e.m.; *p<0.05. (C) cytosolic FUS activates GSK-3 more potently than wild type FUS. Cells were transfected with EGFP control, EGFP-FUS or EGFP-FUS lacking its C-terminal nuclear localization signal (FUSC). Samples were probed on immunoblots for total GSK-3, GSK-3 phosphorylated on serine-9, FUS (using EGFP antibody) and tubulin as a loading control. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N=3, error bars are s.e.m.; **p<0.01, ***p<0.001. (D) cytosolic FUS reduces the VAPB-PTPIP51 interaction more potently than wild type FUS. Cells were transfected with PTPIP51-HA and either EGFP control, EGFP-FUS or EGFP-FUSC. PTPIP51 was immunoprecipitated using the HA tag and bound endogenous VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown. FUS was detected using EGFP antibody. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals. Data were analysed by one-way ANOVA and Tukey’s post-hoc test; N=3. Error bars are s.e.m; **p<0.01, ***p<0.001.
ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria association (2016)
Figure 6

Figure 6. Inhibition of GSK-3 increases ER-mitochondria associations and the VAPB-PTPIP51 interaction.

(A) representative electron micrographs of ER-mitochondria associations in NSC34 cells treated with either vehicle or GSK-3β inhibitors AR-A014418 (1 M) or CT99021 (100 nM) for 16 h. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. N=32 cells and 181-210 mitochondria. Error bars are s.e.m.; *p<0.05. (B) VAPB-PTPIP51 proximity ligation assays of NSC34 cells treated with either vehicle 1 M AR-A014418 or 100 nM CT99021 for 16 h. Cells were also stained for nuclei with DAPI. Bar chart shows relative number of proximity ligation assay signals/cell. Data were analysed by one-way ANOVA and Tukey’s post-hoc test; n=103-173 cells from 5 experiments. Error bars are s.e.m.; **p<0.01, ***p<0.001.
ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria association (2016)
Figure 7

Figure 7. Inhibition of GSK-3 rescues defective ER-mitochondria associations induced by FUS.

Representative electron micrographs of ER-mitochondria associations in NSC34 cells transfected with EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and treated with vehicle or 1 mM AR-A014418 for 16 hours. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. N=26-27 cells and 201-236 mitochondria. Error bars are s.e.m.; *p<0.05, ***p<0.001.
ALS/FTD-associated FUS activates GSK-3β to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria association (2016)
Figure 8

Figure 8. Inhibition of GSK-3 rescues FUS-induced defects in mitochondrial Ca2+ uptake following its release from ER stores.

HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K and then treated with vehicle (DMSO) or 1 M AR-A014418 for 16 hours as indicated. Release of ER Ca2+ was induced by treatment of cells with OxoM. Representative Rhod2 fluorescence traces showing mitochondrial Ca2+ are shown along with bar chart displaying normalized peak values. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N=30-100 cells from 4 experiments. Error bars are s.e.m.; *p<0.05, ***p<0.001.
Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography (2020)
Figure 5

Figure 5: Direct stacking of SAS-6 rings

(A-D) Computationally assembled CrSAS-6[6HR] single (A, C) or double ring in register (ribbon diagram shown in different shades for clarity) (B, D) fitted into the 3D maps of the 36 % T. mirabilis class (A, B) and the 45 % T. agilis class (C, D). Dashed box shows longitudinal section through hub element (left), double chevron viewing point of longitudinal external views (right). Extra unaccounted densities in single ring fitting are indicated by arrowheads (A, C). Note that each elongated hub density can accommodate two tightly stacked CrSAS-6[6HR] rings (B, D), and that only one ring can fit into the thin hub element in the 45% T. agilis class (C, D). Indicated distances stem from measurements on the fitted models.

(E) Purified CrSAS-6[NL] protein self-organized into stacks and analyzed by cryo-ET; one longitudinal and one transverse view are highlighted by a dashed rectangle and a dashed circle, respectively.

(F, G) 2D views through STA of in vitro self-assembled CrSAS-6[NL] proteins. Transverse sections at the indicated heights through one assembly unit (F), as well as longitudinal view (G) of the area delineated by a dashed box in (F); white arrowhead denotes position of line scan along the vertical axis at the level of the hub, with corresponding pixel intensities in arbitrary units (AU). The average distance between two hub elements is 4.5 ± 0.3 nm (N=7; dashed lines).

(H) Longitudinal view of in vitro self-assembled CrSAS-6[NL] proteins STA 3D map. Note densities bridging successive hubs vertically (arrows).

(I) Two CrSAS-6[HR] single rings fitted into the 3D map of in vitro self-assembled CrSAS-6[NL] proteins. Dashed box indicates longitudinal section through hub element (left), double chevron indicates viewing point of longitudinal external view (right). Note that the 3D map can accommodate rings stacked 4.4 nm apart (measured on fitted ring models), and that vertical densities linking hubs are partially occupied.