title: 3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints.
abstract: In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 x 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 A.
authors.affiliation: Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS Université Lyon 1, IFR128 BioSciences Lyon-Gerland, 7, passage du Vercors, 69367 Lyon, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Loquet
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bardiaux
authors.forename: Carole
authors.initials: C
authors.lastname: Gardiennet
authors.forename: Christophe
authors.initials: C
authors.lastname: Blanchet
authors.forename: Marc
authors.initials: M
authors.lastname: Baldus
authors.forename: Michael
authors.initials: M
authors.lastname: Nilges
authors.forename: Thérèse
authors.initials: T
authors.lastname: Malliavin
authors.forename: Anja
authors.initials: A
authors.lastname: Böckmann
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D008958:Models, Molecular; D010750:Phosphoproteins / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012015:Reference Standards
chemical_list: D001426:Bacterial Proteins; C118042:Crh protein, Bacillus subtilis; D010750:Phosphoproteins