The dynamic duo: Combining NMR and small angle scattering in structural biology

Abstract

Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods, and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multidisciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X-ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is well-suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue resolution but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR, crystallography, and SAS is, thus, very useful for analysis of the structure and conformational dynamics of (large) protein complexes in solution. In high molecular weight systems, where NMR data are often sparse, SAS provides additional structural information and can differentiate between NMR-derived models. Scattering data can Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; CSPs, chemical shift perturbations; EM, electron microscopy; IDP, intrinsically disordered proteins; IDR, intrinsically disordered regions; LBT, lanthanide binding tag; NMR, nuclear magnetic resonance spectroscopy; PRE, paramagnetic relaxation enhancement; RDC, residual dipolar coupling; SAS, small angle scattering; SANS, small angle neutron scattering; SAXS, small angle X-ray scattering. also validate the solution conformation of a crystal structure and indicate the presence of conformational equilibria. Here, we review current state-of-the-art approaches for combining NMR, crystallography, and SAS data to characterize protein complexes in solution.

Visualizing the Path of DNA through Proteins Using DREEM Imaging

“…It will be especially useful for characterizing protein-DNA complexes with long length scales and those that result in heterogeneous populations of proteins on the DNA. Furthermore, a growing area in structural biology is the combination of atomic-resolution crystal structures with lower-resolution data from small-angle X-ray scattering, EM, and AFM to generate atomic-level structures of complex assemblies and conformationally flexible proteins (Bustamante et al, 1994; Erie et al, 1994; Griffith, 2013; Griffith and Christiansen, 1978; Hennig and Sattler, 2014; Hura et al, 2013a, 2013b; Janićijević et al, 2003; Lohr et al, 2007; Lyubchenko et al, 2001; Moreno-Herrero et al, 2005; Sanchez et al, 2013; Trinh et al, 2012; Villarreal and Stewart, 2014; Wanner and Schroeder-Reiter, 2008; Williams et al, 2014; Yang et al, 2003; Yeh et al, 2012). DREEM has the capability to significantly increase the constraints on the possible orientations of proteins in multiprotein assemblies on DNA, as demonstrated by our ability to dock the crystal structure of the nucleosome into a subset of the images.…”

“…Finally, the assembly process may occur over long DNA lengths and/or bring distal DNA regions together. An emerging area of structural biology, which is beginning to address this problem, is the combination of high-resolution data from crystallography and NMR with lower-resolution data from techniques such as small-angle X-ray scattering, which provides estimates of the distribution of conformational states (Hennig and Sattler, 2014; Hura et al, 2013a, 2013b; Williams et al, 2014), and electron microscopy (EM) and atomic force microscopy (AFM), which provide images of individual complexes (Bustamante et al, 1994; Erie et al, 1994; Griffith, 2013; Griffith and Christiansen, 1978; Janićijević et al, 2003; Lohr et al, 2007; Lyubchenko et al, 2001; Maletta et al, 2014; Moreno-Herrero et al, 2005; Sanchez et al, 2013; Trinh et al, 2012; Villarreal and Stewart, 2014; Wanner and Schroeder-Reiter, 2008; Yang et al, 2003; Yeh et al, 2012). Although these hybrid methods are promising, a significant limitation to the existing lower-resolution techniques is their limited capability for resolving the location of the nucleic acids within protein-DNA complexes.…”

Structural Analysis of Protein–RNA Complexes in Solution Using NMR Paramagnetic Relaxation Enhancements

“…Additional complementary information can be obtained from combining NMR with small-angle X-ray or neutron scattering (SAXS/SANS) (Bertini et al, 2008;Grishaev, Tugarinov, Kay, Trewhella, & Bax, 2008;Grishaev, Wu, Trewhella, & Bax, 2005;Hennig & Sattler, 2014;Hennig et al, 2013;Huang et al, 2014;Lapinaite et al, 2013;Schwieters et al, 2010) and EPR data (Duss, Michel, et al, 2014;.…”

Small angle neutron scattering contrast variation reveals heterogeneities of interactions in protein gels

“…the shell or the core of core-shell particles 2 , the nanoparticles and polymers in nanocomposites gels 3 , or the network heterogeneities in natural rubber 4 . Contrast variation has also been proven as a powerful technique to study biological structures in dilute regime 5 , including protein-protein, proteinribosome, and protein-DNA complexes 5,6 or larger complexes as caseins 7 . However some precautions must be taken with the use of deuterated solvents since H/D exchange between the labile protons of the scattering objects and the deuterated solvent occurs 8 .…”