High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data

Abstract

X-ray diffraction and Nuclear Magnetic Resonance spectroscopy (NMR) are the staple methods for revealing atomic structures of proteins. Since crystals of biomolecular assemblies and membrane proteins often diffract weakly and such large systems encroach upon the molecular tumbling limit of solution NMR, new methods are essential to extend structures of such systems to high resolution. Here we present a method that incorporates solid-state NMR restraints alongside of X-ray reflections into the conventional model building and refinement steps of structure calculations. Using the 3.7 Å crystal structure of the integral membrane protein complex DsbB-DsbA as a test case yielded a significantly improved backbone precision of 0.92 Å in the transmembrane region, a 58% enhancement from using X-ray reflections alone. Furthermore, addition of solid-state NMR restraints greatly improved the overall quality of the structure by promoting 22% of DsbB transmembrane residues into the most favored regions of Ramachandran space in comparison to the crystal structure. This method is widely applicable to any protein system where X-ray data are available, and is particularly useful for the study of weakly diffracting crystals.

NMR structures of membrane proteins in phospholipid bilayers

“…Subsequently, Bushweller and co-workers have extended the study of DsbB to nanodiscs, which provide a planar bilayer environment amenable to solution NMR studies (Früh et al, 2010). Rienstra and co-workers have utilized MAS solid-state NMR to study the structure of a DsbB-DsbA complex (Tang et al, 2011a) and features of the DsbB protein in endogenous lipid (Tang et al, 2013b). …”

How to tackle protein structural data from solution and solid state: An integrated approach

“…However, such discrepancies may either reflect real differences between the true structures of the molecules in crystals and in solution, or may be due to the different but complementary information contained in the X-ray and NMR data, thus the true structures may be the same but the process of deriving the models by analysing the different types of data may lead to inconsistencies. In a number of cases, refinements performed using combined X-ray and NOE-derived restraints revealed large consistency of the data, resulting in the improvement of the geometry of the model in terms of Ramachandran plot with respect to the structure calculated without NMR data [96][97][98][99][100]. The few violations were interpreted as real differences between the structures in crystals and in solution, mostly ascribable to a limitation of the conformational freedom for some flexible parts and to the presence of crystal packing forces in the solid state.…”

Solid-state NMR: An emerging technique in structural biology of self-assemblies

“…Rienstra, Schwieters et al proposed joint calculation routines [162,163] in XPLOR-NIH simulated annealing [164] to combine ssNMR-based dihedral angle predictions and 13 C-13 C distance restraints with X-ray reflections extracted from crystallographic data in order to improve the precision of the generated ssNMR ensemble, and applied this approach to solve the structure of the membrane protein complex DsbB-DsbA. Similarly, small-angle X-ray scattering (SAXS) data can provide an average volumetric map that is helpful to restraint the conformational space of large protein assemblies.…”