Abstract
The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably `ideal' geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.
Highlights
The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide
The catalytic domain of matrix metalloproteinase 1 (MMP-1), ubiquitin (Ub), the third IgG-binding domain of protein G (GB3), calmodulin (CaM) and CaM bound to the CaM-binding peptide of the death-associated protein kinase (CaM–DAPk), have been refined with REFMAC5 using the structure factors deposited in the PDB for entries 3shi, 3nhe, 1igd, 1exr and 1yr5, respectively
Self-orientation residual dipolar couplings (RDCs) have the advantage with respect to diamagnetic RDCs of depending on the same tensor as pseudo-contact shifts (PCSs): since PCSs are only slightly affected by local mobility and structural inaccuracies, they can provide a robust estimation of this tensor once a structural model is available, so that the RDCs can safely be used for both structural and dynamic analysis
Summary
Long-range paramagnetic NMR data such as pseudo-contact shifts (PCSs; Horrocks & Hall, 1971; Barry et al, 1971; La Mar et al, 1973) and/or self-orientation residual dipolar couplings (RDCs; Tolman et al, 1995; Bothner-By, 1996) arising from a paramagnetic metal coordinated to the protein have been shown to be valuable restraints to help in solving protein structures in the solution state (Gochin & Roder, 1995; Banci et al, 1996, 1998) and since have been thoroughly used (Bertini et al, 2001; Gaponenko et al, 2004; Dıaz-Moreno et al, 2005; Pintacuda et al, 2006; Jensen et al, 2006; Schmitz et al, 2012). PCSs and self-orientation RDCs are here proposed as additional restraints for a joint refinement together with the crystallographic data Owing to their long-range nature, they can be more effective than NOEs as structural restraints and more helpful in disclosing structural differences between the solution and solid states. The joint use of PCS/RDC restraints and X-ray data can indicate differences between solid-state and solution conformations or, in their absence, can be used to refine the protein structure Both PCSs and RDCs can be measured in paramagnetic proteins, and possibly complemented by paramagnetic relaxation enhancements, which can be provided for structural refinement once translated into distance restraints (Bertini et al, 2008). In the case of calmodulin, both free and complexed with a target peptide, the joint refinement does not produce an atomic model fully consistent with both data sets, indicating the possible presence of some structural differences between the protein in solution and in the solid state
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