title: High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5'AMP.
abstract: Native data, anomalous data at three wavelengths and an independent peak-wavelength data set for SeMet-substituted protein have been collected from cryoprotected crystals of the TrpRS-adenylate product (TAM) complex to a resolution limit of 1.7 A. Independent phase sets were developed using SHARP and improved by solvent flipping with SOLOMON using molecular envelopes derived from experimental densities for, respectively, peak-wavelength SAD data from four different crystals, MAD data and their M(S)IRAS combinations with native data. Hendrickson-Lattman phase-probability coefficients from each phase set were used in BUSTER to drive maximum-likelihood refinements of well defined parts of the previously refined room-temperature 2.9 A structure. Maximum-entropy completion followed by manual rebuilding was then used to generate a model for the missing segments, bound ligand and solvent molecules. Surprisingly, peak-wavelength SAD experiments produced the smallest phase errors relative to the refined structures. Selenomethionylated models deviate from one another by 0.25 A and from the native model by 0.38 A, but all have r.m.s. deviations of approximately 1.0 A from the 2.9 A model. Difference Fourier calculations between amplitudes from the 300 K experiment and the new amplitudes at 100 K using 1.7 A model phases show no significant structural changes arising from temperature variation or addition of cryoprotectant. The main differences between low- and high-resolution structures arise from correcting side-chain rotamers in the core of the protein as well as on the surface. These changes improve various structure-validation criteria.
authors.affiliation: Department of Biochemistry and Biophysics, CB# 7260, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: P
authors.initials: P
authors.lastname: Retailleau
authors.forename: Y
authors.initials: Y
authors.lastname: Yin
authors.forename: M
authors.initials: M
authors.lastname: Hu
authors.forename: J
authors.initials: J
authors.lastname: Roach
authors.forename: G
authors.initials: G
authors.lastname: Bricogne
authors.forename: C
authors.initials: C
authors.lastname: Vonrhein
authors.forename: P
authors.initials: P
authors.lastname: Roversi
authors.forename: E
authors.initials: E
authors.lastname: Blanc
authors.forename: R M
authors.initials: RM
authors.lastname: Sweet
authors.forename: C W
authors.initials: CW
authors.lastname: Carter
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008958:Models, Molecular; D008968:Molecular Conformation; D011487:Protein Conformation; D015203:Reproducibility of Results; D012645:Selenomethionine / Q000737:chemistry; D012984:Software; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry*
chemical_list: D012997:Solvents; D012645:Selenomethionine; D014369:Tryptophan-tRNA Ligase