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
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.