Crystal structure of a tRNA acceptor-stem mimic at 1.94 Å resolution

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Phosphorus single-wavelength anomalous diffraction (P-SAD) has theoretical benefits for nucleic acid phasing.
• P-SAD application is hindered by high atomic displacement parameters and poor reflection-to-scatterer ratios.
• AlphaFold3 failed to generate reliable models for the target RNA complex.

Purpose of the Study:

• To determine the crystal structure of a four-stranded RNA complex.
• To investigate the utility of bromine (Br-SAD) and phosphorus (P-SAD) anomalous diffraction for phasing.
• To demonstrate the complementary application of different anomalous signals in RNA crystallography.

Main Methods:

• Collected bromine single-wavelength anomalous diffraction (Br-SAD) data at 0.916 Å on beamline I04.
• Obtained phosphorus anomalous diffraction data at 3.024 Å on beamline I23.
• Utilized experimental phasing with bromine anomalous scattering for structure solution.

Main Results:

• Successfully phased the RNA complex structure using bromine anomalous scattering.
• Phosphorus anomalous peaks corroborated RNA backbone positions and validated the Br-SAD model.
• Direct phasing attempts using phosphorus data failed, aligning with theoretical predictions.
• The final RNA model revealed a pseudo-helical complex stabilized by Watson-Crick and Hoogsteen base pairing.

Conclusions:

• Bromine anomalous diffraction is effective for phasing RNA structures when P-SAD is challenging.
• Complementary use of bromine and phosphorus anomalous signals enhances RNA crystallography.
• The study provides insights into RNA complex structure and crystallization artifacts.