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

Area of Science:

• Structural Biology
• X-ray Crystallography
• Biochemistry

Background:

• The TrpRS-adenylate product (TAM) complex structure was previously refined at room temperature to 2.9 A resolution.
• High-resolution structural data is crucial for understanding enzyme mechanisms and interactions.

Purpose of the Study:

• To determine the high-resolution structure of the TrpRS-adenylate product complex.
• To compare structural data obtained from different experimental phasing methods.
• To investigate the effects of cryoprotection and temperature on protein structure.

Main Methods:

• Collected native, anomalous (three wavelengths), and peak-wavelength data for SeMet-substituted protein crystals.
• Utilized SHARP and SOLOMON for phase determination and refinement.
• Employed BUSTER for maximum-likelihood refinement and maximum-entropy completion.
• Performed difference Fourier analysis to assess structural changes.

Main Results:

• Achieved a resolution limit of 1.7 A for the TrpRS-adenylate product complex.
• Peak-wavelength SAD experiments resulted in the smallest phase errors.
• Selenomethionylated models showed minor deviations (0.25 A) from each other and the native model (0.38 A).
• No significant structural changes were observed due to temperature or cryoprotectant.

Conclusions:

• High-resolution structure determination of the TrpRS-adenylate product complex is feasible using SAD and MAD phasing.
• Peak-wavelength SAD phasing is a reliable method for phase determination.
• The refined structure reveals accurate side-chain rotamer conformations, improving structural validation.