High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5 ' AMP

Citation
P. Retailleau et al., High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5 ' AMP, ACT CRYST D, 57, 2001, pp. 1595-1608
Citations number
63
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Chemistry & Analysis
Journal title
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
ISSN journal
0907-4449 → ACNP
Volume
57
Year of publication
2001
Part
11
Pages
1595 - 1608
Database
ISI
SICI code
0907-4449(200111)57:<1595:HEPFTS>2.0.ZU;2-Z
Abstract
Native data, anomalous data at three wavelengths and an independent peak-wa velength data set for SeMet-substituted protein have been collected from cr yoprotected crystals of the TrpRS-adenylate product (TAM) complex to a reso lution limit of 1.7 Angstrom. Independent phase sets were developed using S HARP and improved by solvent flipping with SOLOMON using molecular envelope s derived from experimental densities for, respectively, peak-wavelength SA D data from four different crystals, MAD data and their M(S)IRAS combinatio ns with native data. Hendrickson-Lattman phase-probability coefficients fro m each phase set were used in BUSTER to drive maximum-likelihood refinement s of well defined parts of the previously refined room-temperature 2.9 Angs trom structure. Maximum-entropy completion followed by manual rebuilding wa s then used to generate a model for the missing segments, bound ligand and solvent molecules. Surprisingly, peak-wavelength SAD experiments produced t he smallest phase errors relative to the refined structures. Selenomethiony lated models deviate from one another by 0.25 Angstrom and from the native model by 0.38 Angstrom, but all have r.m.s. deviations of similar to1.0 Ang strom from the 2.9 Angstrom model. Difference Fourier calculations between amplitudes from the 300 K experiment and the new amplitudes at 100 K using 1.7 Angstrom model phases show no significant structural changes arising fr om temperature variation or addition of cryoprotectant. The main difference s between low- and high-resolution structures arise from correcting side-ch ain rotamers in the core of the protein as well as on the surface. These ch anges improve various structure-validation criteria.