Rapid Determination of Protein Structures in Solution Using NMR Dipolar Couplings

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Once NMR spectra are measured, two main steps follow to determine NMR structure. One is backbone resonance assignment and the other is structure calculation. The both are time consuming steps. We present program MARS and ITAS to speed up NMR structure determination.At first, MARS is robust for automatic backbone resonance assignment of 13C/15N labeled proteins. MARS simultaneously optimizes the local and global quality of assignments in order to minimize the propagation of initial assignment errors and to extract reliable assignments. It works with a wide variety of NMR experiments and is robust against missing chemical shift information. Furthermore, a new method was implemented into MARS, which uses sequential connectivity and experimental residual dipolar couplings (RDCs) simultaneously for NMR resonance assignment when structures are available. Assignment was significantly enhanced when experimental RDCs are additionally matched to back-calculated values from a known three-dimensional structure. The combination of sequential connectivity information with RDC-matching allows for more residues to be assigned reliably and backbone assignments to be more robust against missing data.Secondly, ITAS simultaneously calculates protein structure and assigns the backbone resonances using unassigned chemical shifts and RDCs. Opposite to conventional approaches, where sequential resonance assignment has to be completed prior to structure calculation, partial assignments are used to obtain low-resolution models. These low-resolution models are used to improve the backbone resonance assignment and the improved assignment is again used for structure calculation. Within four to eight iteration steps consisting of automatic assignment using MARS and structure calculation using RosettaNMR a nearly complete resonance assignment and medium accuracy structures of protein backbones are obtained.The automation of resonance assignment allows for significant time savings for resonance assignment compared to manual assignment. Furthermore the ITAS automated structure calculation including automatic resonance assignment without any manual intervention avoids another time consuming step.