Date Published: April 01, 2016
Publisher: International Union of Crystallography
Author(s): Blaine H. M. Mooers.
The crystal structure of a 32-base-pair RNA double helix (675 non-H atoms) from a trypanosome RNA-editing substrate was determined with 1.05 Å resolution X-ray diffraction data starting from random phases using the direct-methods computer program SIR2014. Success was achieved in the presence of two levels of translational pseudosymmetry caused by three helical repeats.
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 ▸). These constraints are relaxed when calcium or heavier atoms are present, when Patterson superposition methods are used or when Patterson methods and heavy atoms are used together (Burla et al., 2006 ▸; Caliandro et al., 2007 ▸, 2008 ▸; Mooers & Matthews, 2004 ▸, 2006 ▸). We found only one report of ab initio direct methods being successfully applied to an unknown RNA molecule (Safaee et al., 2013 ▸). 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 ▸). On the other hand, 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 molecular-replacement structure determination of proteins (Dauter et al., 2005 ▸). The role of TPS in phasing has been explored many times in chemical crystallography and is a current interest in biological crystallography (Hauptman & Karle, 1959 ▸; Böhme, 1982 ▸; Gramlich, 1984 ▸; Cascarano et al., 1985 ▸, 1987 ▸; Fan, Qian et al., 1988 ▸; Fan, Yao et al., 1988 ▸; Zwart et al., 2008 ▸; Read et al., 2013 ▸). The rational dependence of the atoms related by TPS leads to sets of strong reflections and weak reflections. Most of the phase relationships depend on strong reflections if the presence of TPS is ignored. The weak reflections can be used to form separate phase relationships (Cascarano et al., 1988a ▸,b ▸; Fan, Qian et al., 1988 ▸; Fan, Yao et al., 1988 ▸). Rotational pseudosymmetry in crystal structures of short dsRNAs has been reported (Kondo et al., 2008 ▸) and the prospects for direct methods with oligonucleotides shorter than one helical turn have been explored (Hubbard et al., 1994 ▸), but we know of no published applications of direct methods to nucleic acids with TPS present. The most common TPS in protein crystals involves two molecules in the asymmetric unit. Sometimes TPS is found within a single protein; three of 1007 protein superfamilies have internal TPS (Myers-Turnbull et al., 2014 ▸). In contrast, RNA double helices longer than one helical turn could have imperfect TPS caused by the helical repeats. This TPS could restrict success in direct methods to RNAs of one helical turn in length or shorter. Previous nucleic acid structures determined ab initio by direct methods have been one helical turn long or shorter (Egli et al., 1998 ▸; Han, 2001 ▸; Safaee et al., 2013 ▸; Luo et al., 2014 ▸).
We compared the structure determinations of dsRNA with three helical turns and of a hairpin with one helical turn and thus no TPS. The diffraction data for the dsRNA were 99% complete (Table 1 ▸) and had a resolution limit of 1.05 Å (Fig. 1 ▸a). The native Patterson map showed evidence of TPS (Fig. 2 ▸). The hairpin RNA was the closest in size to the 32 nt RNA of the available RNA structures with diffraction data at similar resolution. Its diffraction data were nearly complete, and the structure lacked calcium or heavier atoms. Next, we describe the initial structure determination of the dsRNA. The same structure-determination procedure was used with the data from the 27 nt hairpin RNA. We compared the distribution of the number of failed trials before a correct structure for the dsRNA and the hairpin, and found a large difference. We also found differences in the distributions of the intensities and of the vectors used to shift misplaced trial structures. In addition, we found a difference in the sensitivity to the removal of the strongest reflections. The details of the structure of the dsRNA are irrelevant to the central question of this paper and will be described elsewhere. Because each case has a sample size of one, the results reported below cannot be used to make inferences about the ease of structure determination by direct methods with diffraction data from other RNAs.