Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

Abstract

We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.

Fig. 1.

Flow chart illustrating the new NMR structure determination protocol. Expert NMR data analysis is shown as rounded rectangles, whereas structural modeling based on interpreted data (such as chemical shifts, peak lists, and RDCs) is in boxed shapes.

Fig. 2.

RASREC Rosetta results for maltose-binding protein. The calculations used experimental data collected in this study (ER690 ILV sample). Shown are structural superpositions of the RASREC CS-Rosetta structure (red) with the X-ray crystal structure of the holo protein, 1ez9 (blue). (A) Histogram of the opening angles of the 50 lowest-energy structures. (B) The RASREC CS-Rosetta structure with the smallest opening angle within the 10 lowest by Rosetta energy is superimposed on the most-closed crystal structure (3 mbp). Because the opening angle is heterogeneous (see A), we show in C and D superpositions of the N- and C-terminal domains, respectively, for the structure which best fits the RDC data among the 10 lowest by Rosetta energy.

Fig. 3.

RASREC CS-Rosetta results for five target proteins: (A) sensory rhodopsin, (B) BamC N-terminal domain, (C) BamC C-terminal domain, (D) SgR145, and (E) WR73. Rosetta structures (red) are superimposed with reference structures (blue). Each sub-figure shows the cartoon representation of the lowest-energy structure (Left) and close up of the core region that illustrates accuracy of side chains (Right). Protein Data Bank–accession codes of the X-ray reference structures in A–E are 1h68, 2yh6, 2yh5, 3mer, and 2loy, respectively.