High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5'AMP
Retailleau, P.; Yin, Y.; Hu, M.; Roach, J.; Bricogne, G.; Vonrhein, C.; Roversi, P.; Blanc, E.; Sweet, R.M.; Carter, C.W.
Acta Crystallographica. Section D Biological Crystallography 57(Pt 11): 1595-1608
2001
ISSN/ISBN: 0907-4449 PMID: 11679724 DOI: 10.1107/s090744490101215x
Accession: 010753305
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 ANG. 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 ANG 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 ANG and from the native model by 0.38 ANG, but all have r.m.s. deviations of apprx1.0 ANG from the 2.9 ANG model. Difference Fourier calculations between amplitudes from the 300 K experiment and the new amplitudes at 100 K using 1.7 ANG 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.