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. 2018 Oct 2;115(42):E9782–E9791. doi: 10.1073/pnas.1806174115

Fig. 4.

Fig. 4.

Structural measurements on mCherry-hnRNPA2-LC polymers. (A) Predictions of backbone ϕ/ψ torsion angles by TALOS-N, based on assigned solid-state NMR chemical shifts. Error bars are uncertainties reported by TALOS-N. Filled and open symbols represent “strong” and “generous” predictions, respectively. Black bars indicate regions where solid-state NMR signals are assigned. (B) Normalized 15N-BARE decay curves for residues N289–F291. Error bars represent the rms noise in the NMR spectrum and dashed lines are Gaussian fits to the data. (C) 13C-13C PITHIRDS-CT dipolar recoupling data for mCherry-hnRNPA2-LC polymers that were 13C-labeled only at backbone carbonyl sites of Tyr residues within residues 280–341. Red diamonds are normalized peak intensities. Lines are simulated data for linear chains of 13C labels with the indicated interatomic spacings. Error due to noise is smaller than the symbol size. (D) Dark-field TEM image of unstained mCherry-hnRNPA2-LC polymers. Yellow and cyan arrows indicate mCherry-hnRNPA2-LC polymers and TMV rods, respectively. Examples of 60- × 80-nm integration boxes used in the MPL measurement are shown. (E) Histogram of apparent MPL values. The red line is a single Gaussian fit to the data, centered at 86.0 ± 0.6 kDa/nm with a full-width half-maximum value of 22.0 ± 0.9 kDa/nm. The vertical gray dashed line is the expected MPL value for cross-β polymers with an intermolecular spacing of 0.48 nm and with the mCherry-hnRNPA2-LC monomer molecular weight of 45.8 kDa. The vertical black dashed line is for an average molecular weight of 40.9 kDa, corresponding to partial cleavage of the mCherry tag for 45% of the monomers. (F) Histogram of apparent MPL values for mCherry-hnRNPA2-LC D290V mutant polymers. The red line is a Gaussian fit to the data, centered at 95.8 ± 1.0 kDa/nm with a full-width half-maximum value of 28.1 ± 1.4 kDa/nm. The vertical black dashed line is the expected value for cross-β polymers with an intermolecular spacing of 0.48 nm and the mCherry-hnRNPA2-LC D290V molecular weight of 46.2 kDa.