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1 Supplemental Information for: New Insight into the Structure of RNA in Red clover necrotic mosaic virus and the Role of Divalent Cations Revealed by Small-Angle Neutron Scattering Stanton L. Martin a, *, Lilin He b,c, *, Flora Meilleur c,d, Richard H. Guenther a, Tim L. Sit a, Steven A. Lommel a, William T. Heller b,c, ** a Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695; b Center for Structural Molecular Biology and Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831; c Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831; d Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina *Stanton L Martin and Lilin He contributed equally to the work. **Corresponding author: William T. Heller Oak Ridge National Laboratory Neutron Scattering Sciences Division PO Box 2008; MS 6473 Oak Ridge, TN37831 Phone: Fax: hellerwt@ornl.gov 1
2 SANS Data Analysis. The radius of gyration, R g, and the forward scatter, I(0), were determined using the Guinier approximation [1] from the slope of a straight line fit to the data transformed to I q Ln vs. 2 q, which was accomplished using the software implemented by NIST in IGOR Pro 6.1 [2]. The Guinier plots and associated fit lines are shown in Figure S1, while the R g and I(0) are presented in Table S1. The slight upturn at the lowest q-values shown suggest that a high molecular weight contaminant was present. As a result, the first three points were omitted for the fitting. The distance distribution, P(r), which is related to the SANS intensity by a Fourier transform, was calculated using the program GNOM [3]. The radial scattering density distribution function, r s, where s is the SLD for the solvent, was derived from the SANS data at each contrast value as described previously [4]. The maximum linear dimension determined during the P(r) fitting was 350 Å, which agrees well with the 366 Å RCNMV capsid diameter determined by cryo-em [5]. The position of the maxima of the 63%, 77%, and 81% D 2 O data sets are at ~220 Å and the shapes of the P(r) curves are consistent. The most probable distance occurs at a position greater than half of D max, indicating that the inner core of the virion is essentially free of protein and RNA. The 59% D 2 O curve, which is the only dataset collected between the contrast match-points of the protein and RNA, peaks at ~250Å, which results from the effect of the destructive interference on the SANS data at this specific contrast condition and is not a sign of an inconsistent data set. 2
3 Figure S1. Guinier plots of SANS CV data sets collected at 59% D 2 O ( ), 63% D 2 O ( ), 77% D 2 O ( ) and 81% D 2 O ( ). The curves have been offset for clarity. The first three points were omitted from the Guinier fitting to reduce the effect of any high-molecular weight contaminants on the fits. The two sets of SANS CV data collected for each sample at the two different sampleto-detector distances were fit simultaneously using the 4-shell model described in Methods (the solid lines). 3
4 Table S1. The radius of gyration, R g, and zero angle intensity, I(0), of wild type RCNMV particles derived from Guinier Approximation [1]. D 2 O R g (Å) I(0) (cm -1 ) 59% ± ± % ± ± % ± ± % ± ±0.06 4
5 Figure S2. Distance distribution functions, P(r), determined from the RCNMV SANS CV data collected at 59% D 2 O ( ), 63% D 2 O ( ), 77% D 2 O( ) and 81% D 2 O ( ) using the GNOM program [3]. The maxima of all curves were normalized to unity. 5
6 Table S2. The 4-shell model fitting parameters for the RCNMV virion determined from the SANS CV data. Shell Thickness (Å) SLD ( 10-6 Å -2 ) 59% D 2 O 63% D 2 O 77% D 2 O 81% D 2 O C 87.95± ± ± ± ±0.01 R 31.75± ± ± ± ±0.01 S 33.30± ± ± ± ±0.01 P 28.65± ± ± ± ±0.01 Solvent
7 References: [1] A. Guinier, G. Fournet, Small-angle Scattering of X-rays, Wiley, New York, [2] S.R. Kline, J. Appl. Crystallogr. 39 (2006) [3] A.V. Semenyuk, D.I. Svergun, J. Appl. Crystallogr. 24 (1991) [4] L.L. He, A. Piper, F. Meilleur, D.A.A. Myles, R. Hernandez, D.T. Brown, W.T. Heller, J. Virol. 84 (2010) [5] M.B. Sherman, R.H. Guenther, F. Tama, T.L. Sit, C.L. Brooks, A.M. Mikhailov, E.V. Orlova, T.S. Baker, S.A. Lommel, J. Virol. 80 (2006)
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