RAVEN: AUTOMATION OF SOLID STATE NMR SPECTRAL CROSS-PEAKS ASSIGNMENT FOR THE ANALYSIS OF MACROMOLECULES STRUCTURE AND DYNAMICS

The determination of 3D structures of macromolecules by magic angle spinning solid state NMR (MAS ssNMR) is considered a long and complex process.

A new user-friendly software named Raven is presented, with the ability to analyze spectral ssNMR cross-peaks automatically using a preliminary assignment, find structural constrains and produce input for external software of structure and dynamics calculations.

Raven offers a full control in getting the desirable and suitable results by multiple tools and features such as a custom tool for determination of isotope labeling, the ability to control the probability function and range for matching correlations, the extent of ambiguous cross peaks assignment and more. Hence, it saves a significant amount of time.

Three features of Raven are demonstrated: (i) The cross-peaks resulting from a 2D ¹³C-¹³C correlation spectrum of the T2Q mutant of the protein GB1 were automatically matched by Raven and classified according to their ambiguity and to their properties – short, medium and long-range; (ii) The intensities of specific correlations in the spectra of the M13 bacteriophage virus were sampled in different mixing times using the UCSF Analysis tool of Raven and the results gave indications to the rigid and dynamic regions of the virus capsid; (iii) Another feature of the program predicts the level of expected ambiguity when probing a chosen macromolecule by ssNMR. Using this tool, we determined the number of expected non-ambiguous signals in a 2D spectrum of the capsid of the spherical phage MS2 using chemical shifts produced by ShiftX and the current model of the MS2 virus (pdb id 2MS2). This tool allows a reliable prediction of spectral complexity and will aid in designing particular labeling schemes.