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To obtain distance restraints from NOES, N-edited NOESY and C-edited NOESY spectra on aliphatic and aromatic groups were acquired on a 1 mM, C, N-WT-ACD sample in NMR buffer (50 mM sodium phosphate, pH 7.5100 mM NaCl, 0.1 mM EDTA, and 1 mM PMSF).
We discuss approaches for the design of RNA-protein complexes for NMR structural studies, established and emerging isotope and segmental labeling schemes suitable for large RNPs and how to gain distance restraints from NOEs, PREs and EPR and orientational information from RDCs and SAXS/SANS in such systems.
The target function comprised a potential function for experimental distance restraints (e.g. obtained from NOEs or CHHC correlations), a quadratic van der Waals repulsion term, a square-well potential for torsion angles and a torsion angle database potential of mean force.
Adjacent canonical base pairs and therefore potential helices can be identified from NOEs between imino protons separated by <5 Å.
The imino protons were assigned from NOEs between adjacent base pairs and between their corresponding base-paired N-dC amino and H2-dA protons.
These were started from the equilibrated structures, and simulated without any restraints from NOEs or for hydrogen bonds, by using constant pressure and temperature.
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