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

P. Retailleau, Yuhui Yin, M. Hu, J. Roach, G. Bricogne, C. Vonrhein, P. Roversi, E. Blanc, R. M. Sweet, Jr Carter C.W.

Research output: Contribution to journalArticle

25 Citations (Scopus)

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 Å. 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 Å 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 Å and from the native model by 0.38 Å, but all have r.m.s. deviations of ∼1.0 Å from the 2.9 Å model. Difference Fourier calculations between amplitudes from the 300 K experiment and the new amplitudes at 100 K using 1.7 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.

Original languageEnglish (US)
Pages (from-to)1595-1608
Number of pages14
JournalActa Crystallographica Section D: Biological Crystallography
Volume57
Issue number11
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Tryptophan-tRNA Ligase
Temperature
high resolution
Entropy
Wavelength
Proteins
wavelengths
Ligands
proteins
Crystals
phase error
Infrared Astronomy Satellite
Maximum likelihood
crystals
envelopes
Experiments
entropy
deviation
ligands
Molecules

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biophysics
  • Condensed Matter Physics
  • Structural Biology

Cite this

High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5′AMP. / Retailleau, P.; Yin, Yuhui; Hu, M.; Roach, J.; Bricogne, G.; Vonrhein, C.; Roversi, P.; Blanc, E.; Sweet, R. M.; Carter C.W., Jr.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 57, No. 11, 2001, p. 1595-1608.

Research output: Contribution to journalArticle

Retailleau, P, Yin, Y, Hu, M, Roach, J, Bricogne, G, Vonrhein, C, Roversi, P, Blanc, E, Sweet, RM & Carter C.W., J 2001, 'High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5′AMP', Acta Crystallographica Section D: Biological Crystallography, vol. 57, no. 11, pp. 1595-1608. https://doi.org/10.1107/S090744490101215X
Retailleau, P. ; Yin, Yuhui ; Hu, M. ; Roach, J. ; Bricogne, G. ; Vonrhein, C. ; Roversi, P. ; Blanc, E. ; Sweet, R. M. ; Carter C.W., Jr. / High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5′AMP. In: Acta Crystallographica Section D: Biological Crystallography. 2001 ; Vol. 57, No. 11. pp. 1595-1608.
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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 {\AA}. 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 {\AA} 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 {\AA} and from the native model by 0.38 {\AA}, but all have r.m.s. deviations of ∼1.0 {\AA} from the 2.9 {\AA} model. Difference Fourier calculations between amplitudes from the 300 K experiment and the new amplitudes at 100 K using 1.7 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.",
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AU - Yin, Yuhui

AU - Hu, M.

AU - Roach, J.

AU - Bricogne, G.

AU - Vonrhein, C.

AU - Roversi, P.

AU - Blanc, E.

AU - Sweet, R. M.

AU - Carter C.W., Jr

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