Direct-methods structure determination of a trypanosome RNA-editing substrate fragment with translational pseudosymmetry.

Blaine H M Mooers

doi:10.1107/s2059798316001224

Abstract

Using direct methods starting from random phases, the crystal structure of a 32-base-pair RNA (675 non-H RNA atoms in the asymmetric unit) was determined using only the native diffraction data (resolution limit 1.05 Å) and the computer program SIR2014. The almost three helical turns of the RNA in the asymmetric unit introduced partial or imperfect translational pseudosymmetry (TPS) that modulated the intensities when averaged by the l Miller indices but still escaped automated detection. Almost six times as many random phase sets had to be tested on average to reach a correct structure compared with a similar-sized RNA hairpin (27 nucleotides, 580 non-H RNA atoms) without TPS. More sensitive methods are needed for the automated detection of partial TPS.

Highlights

Automated structure determinations by direct methods of equal-atom proteins with 1000 non-H protein atoms have been achieved when starting from random phase angles, when using dual-space methods and when the diffraction data extend to atomic resolution (Sheldrick, 1990; Morris & Bricogne, 2003; Langs & Hauptman, 2011; Giacovazzo, 1998, 2014)
Success in direct-methods structure determination could be expected to be easier with nucleic acids than with proteins because the P atoms in the backbone of RNA are electron-dense, even though they are sometimes in two alternate conformations (Luo et al, 2014), and because the P atoms occur at a higher frequency ($1 in 20) in nucleic acids than S atoms occur in proteins (1 in 100–300; Ramagopal et al, 2003)
We compared the distribution of the number of failed trials before a correct structure for the double-stranded RNA (dsRNA) and the hairpin, and found a large difference

Summary

Introduction

Automated structure determinations by direct methods of equal-atom proteins (i.e. atoms lighter than calcium) with 1000 non-H protein atoms have been achieved when starting from random phase angles (i.e. ab initio direct methods), when using dual-space methods and when the diffraction data extend to atomic resolution (Sheldrick, 1990; Morris & Bricogne, 2003; Langs & Hauptman, 2011; Giacovazzo, 1998, 2014). Translational pseudosymmetry (TPS) caused by helices longer than one turn may inhibit structure determination by direct methods because the internal symmetry violates the assumption that the atoms in the asymmetric unit are randomly distributed. This idea is supported by many reports of TPS hindering the direct-methods structure determination of small-molecule crystal structures and the molecularreplacement structure determination of proteins

Methods

Results

Conclusion