Volume 101, Issue 1, Pages 167-175 (July 2011) Ensemble of Secondary Structures for Encapsidated Satellite Tobacco Mosaic Virus RNA Consistent with Chemical Probing and Crystallography Constraints  Susan J. Schroeder, Jonathan W. Stone, Samuel Bleckley, Theodore Gibbons, Deborah M. Mathews  Biophysical Journal  Volume 101, Issue 1, Pages 167-175 (July 2011) DOI: 10.1016/j.bpj.2011.05.053 Copyright © 2011 Biophysical Society Terms and Conditions

Figure 1 Model of electron density for RNA helices in the 1.8 Ǻ crystal structure of STMV virus particles from the VIPER database (Protein Data Bank 1A34) (3,5). The length of the RNA helix shown in yellow is 36.5 Ǻ. Biophysical Journal 2011 101, 167-175DOI: (10.1016/j.bpj.2011.05.053) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 2 Experimental constraints define a region of the RNA folding funnel. Each smaller gray cone represents a filter based on experimental data such as chemical modification, the minimum number and lengths of helices, local pairing, or no lonely pairs. The intersection of all the filters is represented in red. Biophysical Journal 2011 101, 167-175DOI: (10.1016/j.bpj.2011.05.053) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 3 Hairpins with the best score for each length in every 30-nucleotide window are shown as bars with a length corresponding to the number of nucleotides used to form the hairpin. The color of the bar represents the score for the hairpin calculated using Eq. 1. All of these hairpins are the input for the Assembly algorithm. Biophysical Journal 2011 101, 167-175DOI: (10.1016/j.bpj.2011.05.053) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 4 Best representative 30 hairpins from the ensemble of STMV RNA secondary structures generated by the Crumple and Sliding Windows and Assembly algorithms. Red dots indicate nucleotides chemically modified by DMS, kethoxal, or CMCT. No constraints or interpretation is applied to nucleotides protected from chemical modification because there are many sources of protection from chemical modification, including basepairing, RNA tertiary interaction, protein-RNA interactions, and slower diffusion into the center of the virus capsid. The figure emphasizes the 30 hairpins that are consistent with the crystallographic observations, chemical modification, and the model of cotranscriptional folding and assembly. Other long-range basepairing interactions are possible between nucleotides outside of the 30 hairpins shown, but no attempt is made to predict further secondary structure elements. Primer extension reactions begin reporting chemical modification at nucleotide 970. Green nucleotides are sites of natural variation in STMV RNA (39–41). Tick marks indicate every 10 nucleotides. Biophysical Journal 2011 101, 167-175DOI: (10.1016/j.bpj.2011.05.053) Copyright © 2011 Biophysical Society Terms and Conditions