Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) Size exclusion chromatogram of 3a expressed in insect cells and extracted and purified in DDM (left). Pooled fractions corresponding to dimeric 3a are highlighted in blue. Coomassie-stained SDS-PAGE of pooled dimeric 3a-containing fractions (center) and of 3a following reconstitution into PC lipids (right). This is a representative preparation used for 3a proteoliposome experiments. WT 3a was purified into DDM six times with similar biochemical behavior. Proteoliposomes from three Soy PC preparations were used for activity assays. (b) Size exclusion chromatogram of dimeric 3a reconstituted into MSP1E3D1 lipid nanodiscs (left). Pooled fractions are highlighted blue. 3a was incorporated into nanodiscs three separate times with similar biochemical behavior, and this representative preparation was used for dimeric 3a cryo-EM data collection (c,d) Same as (a,b), but for tetrameric 3a. The tetrameric peak has been observed in all WT 3a preparations. This representative preparation was used for cryo-EM of tetrameric 3a. (e) GFP fluorescence chromatogram of 3a expressed in SF9 cells and extracted in DDM detergent. Samples were run on a Superose 6 column.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Purification, Staining, SDS Page, Activity Assay, Cryo-EM Sample Prep, Fluorescence

Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) Local resolution estimated in Relion colored as indicated on the final map. (b) Angular distribution of particles used in final refinement with final map for reference. (c) Fourier Shell Correlation (FSC) relationships (masked) between (black) the two unfiltered half-maps from refinement and used for calculating overall resolution at 0.143, (red) the final map and model, (gray) half-map one and model, and (blue) half-map and model.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Cryo-EM Sample Prep

Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) Cryo-EM map of the 3a dimer in MSP1E3D1 nanodiscs at 2.1 Å nominal resolution viewed from the membrane plane. One subunit is colored gray and the second subunit is colored with transmembrane region orange and cytosolic domain (CD) pink. Density from the nanodisc MSP1E3D1 is colored tan and the DOPE lipid is coral. (b,c) Model of dimeric 3a viewed (b) from the membrane (as in (a)) and (c) from the extracellular or lumenal side. (d) Zoomed in view of the interaction between subunits in the CD with residues forming the hydrophobic core indicated. (e) Schematic of a 3a monomer. Secondary structure elements are indicated and unmodeled termini and a 5 amino acid β3-β4 loop are shown with dashed lines. (f) Cryo-EM density and model for a selected strand from the CD at two different thresholds. (g) Zoomed-in view of density in the MSP1E3D1 interaction region. (h) Model in the same region as (g) with key residues in the area displayed as sticks.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Cryo-EM Sample Prep

Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) Local resolution estimated in cryoSPARC at the indicated FSC thresholds colored as indicated on the density modified map. (b) Angular distribution of particles used in final refinement with map for reference. (c) ResLog analysis conducted in cryoSPARC at the indicated FSC thresholds. (d) Fourier Shell Correlation as calculated in cryoSPARC before (top) and after (bottom) mask auto-tightening in the final round of refinement. (e) Fourier Shell Correlation (FSC) relationships (masked) calculated in Phenix between (black) the two unfiltered half-maps from refinement and used for calculating overall resolution at 0.143, (red) the final map and model, (gray) half-map one and model, and (blue) half-map and model. (f) Comparison of the original (2.9 Å) 3a map left to the high-resolution (2.1 Å) map before (middle) and after (right) Phenix density modification.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Cryo-EM Sample Prep, Modification

Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) Local resolution estimated in Relion colored as indicated on the final map. (b) Angular distribution of particles used in final refinement with final map for reference. (c) Fourier Shell Correlation (FSC) relationships (unmasked) between (black) the two unfiltered half-maps from refinement and used for calculating overall resolution at 0.143, (red) the final map and model, (gray) half-map one and model, and (blue) half-map and model.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Cryo-EM Sample Prep

Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) View of a 3a dimer from the membrane plane with key regions labeled and with planes (dotted lines) and viewing direction (arrow) for (g-i) indicated. (b) The 3a upper tunnel with solvent-excluded surface shown for each subunit and water molecules shown as light blue spheres. The approximate lipid bilayer region is marked by black lines. One 3a subunit is colored orange and the other in gray. (c) (Left) Model of 3a (gray) with the HOLE path through the upper tunnel shown in transparent cyan. Lipid (coral) is displayed as sticks. (Middle) Radius of the upper tunnel as a function of distance from the cavity center calculated with HOLE. (Right) View into the upper tunnel from the membrane with key residues shown as sticks. (d) As in (b), for a view of the lower tunnel. (e) As in (c), for the lower tunnel, with the view on the right into the tunnel from the cytosol. (f) The intersubunit tunnel with bound DOPE lipid (coral) shown as space-filling spheres. (Right-top) Zoomed-in view of the lipid interaction with key residues shown as sticks. (Right-bottom) Cut-through view of the cryo-EM density of the lipid interaction region viewed from the cytosolic side. (g-i) Model and transparent surface of the viewing planes indicated in (a). Water molecules are shown as light blue spheres. One 3a subunit is colored orange and the other in gray. (j) The solvent excluded surface of 3a colored from hydrophilic (dark cyan) to most hydrophobic (dark orange). A membrane-facing hydrophilic groove is outlined. (k) Key residues in the hydrophilic groove are depicted as sticks colored in dark cyan.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Labeling, Cryo-EM Sample Prep

Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) Two-dimensional class average of tetrameric 3a in MSP1E3D1 lipid nanodiscs (above) and cryo-EM map at 6.5 Å nominal resolution (lower). (b) Approximate assembly of 3a tetramers. Two 3a dimer structures are rigid-body fit into the tetrameric cryo-EM map. (c) Zoomed in view of the interface between 3a dimers observed after rigid-body fitting with residues potentially mediating the interaction displayed as sticks.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Cryo-EM Sample Prep

Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) 3a-GFP fluorescence localization in HEK293 cells transfected with 3a-EGFP or 3aΔN-EGFP. Single plane and brightest-point projection are displayed for each. Scale bar, 10 μm. (b) Side view and view from the extracellular/lumenal space for dimeric 3a cryo-EM density (gray) from the original (left) and high-resolution (right) maps with unmodeled extended density above the mouth of the pore that may correspond to the N-terminal regions colored in purple. A 3a dimer model is drawn orange (TMD) and pink (CD) inside the density. The position of the final modeled N-terminal residue (S40) is indicated. (c) 3a-EGFP or 3aΔN-EGFP field of view with multiple cells imaged using a 20X objective. Scale bar, 20 μm. (d) Additional images of cells imaged with the 63X objective with both single plane and brightest-point projections displayed. Scale bar, 10 μm.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Fluorescence, Transfection, Cryo-EM Sample Prep

Journal: Nature structural & molecular biology

Article Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

doi: 10.1038/s41594-021-00619-0

Figure Lengend Snippet: (a) SDS-PAGE gels with 3a and mock liposome samples prior to (left) and post (right) gel slice extraction for mass spectrometry. Approximate outline of the extracted region is shown with a dotted line. The control and 3a samples were from a liposome preparation used for both electrophysiological and fluorescence assays. (b) Same as (a), but for 3a in DDM sample (prior to liposome incorporation). This sample was from a DDM prep used for both cryo-EM and proteoliposome preparations. (c) Summary table of mass spectrometry analysis for the three analyzed samples. The only other significant hits were to human keratin proteins from the common contaminant library. No additional hits were detected from the G. max (soy) search.

Article Snippet: Consistently, in a cryo-EM structure of dimeric 3a determined in the presence of 100 μM emodin to 3.7 Å resolution ( and , Supplemental Data Fig. 3 , ), we observed no evidence of emodin binding or significant structural changes compared to apo-3a.

Techniques: Mass Spectrometry, SDS Page, Fluorescence, Cryo-EM Sample Prep

Journal: Antioxidants

Article Title: 2-Methoxystypandrone from Polygonum cuspidatum Rejuvenates Senescence by Reducing Mitochondrial ROS

doi: 10.3390/antiox15030357

Figure Lengend Snippet: 2-methoxystypandrone (2-MS) inhibits mitochondrial ROS production in senescent fibroblasts. ( A ) Senescent fibroblasts treated with DMSO (0.01%), emodin-1-O-β-glucoside (8 μM), emodin-8-glucoside (8 μM), 2-methoxystypandrone (2-MS) (8 μM), or polydatin (8 μM) for 12 days at 4-day intervals. Mitochondrial ROS were measured after staining cells with 30 µM dihydrorhodamine 123 (DHR123. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post–hoc test, with results considered not significant (n.s.) or significant at ** p < 0.01. Data represent the mean ± S.D., n = 3. ( B ) Senescent fibroblasts treated with DMSO (0.01%) or 2-MS (4, 8, and 12 μM) for 12 days at 4-day intervals. Mitochondrial ROS were measured after staining cells with 30 µM DHR123. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post–hoc test, with results considered not significant (n.s.) or significant at ** p < 0.01. Data represent the mean ± S.D., n = 3. ( C ) Senescent fibroblasts treated with DMSO (0.01%) or 2-MS (4, 8, and 12 μM) for 12 days at 4-day intervals. DNA tail length was measured using the neutral comet assay. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post–hoc test, with results considered significant at ** p < 0.01. For each condition, the experiment was repeated three times, yielding three independent sets of data (biological triplicates). Sixteen different samples were analyzed to generate each biological triplicate. Data represent the mean ± S.D.

Article Snippet: Mitochondrial ROS levels were measured in senescent fibroblasts treated with DMSO (D8418; Sigma, St. Louis, MO, USA, 0.01%), emodin-1-O-β-glucoside ( CFN93238 ; ChemFaces, Wuhan, China, 8 μM), emodin-8-glucoside (HY-N0311; MedChemExpress, Monmouth, NJ, USA, 8 μM), 2-MS ( CFN97412 ; ChemFaces, 8 μM), or polydatin (15721-25G; Sigma, 8 μM) for 12 days at 4-day intervals.

Techniques: Staining, Neutral Comet Assay