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. 2007 Jul;15(7):761-72.
doi: 10.1016/j.str.2007.06.003.

A general strategy to solve the phase problem in RNA crystallography

Amanda Y Keel  1 , Robert P Rambo, Robert T Batey, Jeffrey S Kieft
Affiliations

A general strategy to solve the phase problem in RNA crystallography

Amanda Y Keel et al. Structure. 2007 Jul.

Abstract

X-ray crystallography of biologically important RNA molecules has been hampered by technical challenges, including finding heavy-atom derivatives to obtain high-quality experimental phase information. Existing techniques have drawbacks, limiting the rate at which important new structures are solved. To address this, we have developed a reliable means to localize heavy atoms specifically to virtually any RNA. By solving the crystal structures of thirteen variants of the G*U wobble pair cation binding motif, we have identified a version that when inserted into an RNA helix introduces a high-occupancy cation binding site suitable for phasing. This "directed soaking" strategy can be integrated fully into existing RNA crystallography methods, potentially increasing the rate at which important structures are solved and facilitating routine solving of structures using Cu-Kalpha radiation. This method already has been used to solve several crystal structures.

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Figures

Figure 1
Figure 1. The use of G·U pairs and the crystallization chassis
a) Schematic of a canonical RNA G-C base-pair and a G·U wobble pair. The G·U pair places partially negative charges in the major groove, forming a pocket for cation binding. b) Diagram of intermolecular packing in the crystal of the SRP RNA-M domain previously reported by Batey et al (Batey et al., 2000; Batey et al., 2001). The end of RNA helix P1 (black) stacks on P1 from an adjacent molecule (grey) in a fairly loose intermolecular contact (boxed). No other crystal contacts are made by this portion of the RNA. c) The structure of the SRP RNA-M domain complex is shown on the left with the mutated helix. In the middle is a schematic of this complex. The protein binding site is shown in grey and the hashed box denotes the portion involved in the majority of crystal contacts. The solid box is the portion of the P1 helix that is displayed by this crystallization chassis, and which was mutated into the various sequences shown at right.
Figure 2
Figure 2. Engineered cation binding
a) Electron density and structure of the wild-type SRP RNA-M domain complex, showing the portion of the helix that was varied. The view is into the major groove. Two cobalt (III) hexammine ions (magenta) are located near the phosphate backbone (upper right site) and two adjacent G-C pairs (lower left). b) Electron density and structure of the variant PM04, with the tandem wobble pairs shown in cyan and the resultant major groove-bound cobalt (III) hexammine in magenta. c) Anomalous difference Fourier map (contoured at 7 σ in red) of PM04 superimposed on the structure. The tandem wobble pairs are shown in cyan. The endogenous reference site is at the top, and the new engineered site at the bottom. d) Comparison of the very well-ordered reference site to the new site in PM04 in a 2Fo-Fc map, at 2 Å resolution, contoured at 2 σ.
Figure 3
Figure 3. Comparison of cation binding in selected variants of the motif
a) Comparison of cobalt (III) hexammine binding in the major groove of three representative motif variants. Major groove carbonyls of the wobble G·U pairs and the flanking Watson-Crick pairs are shown in red, N7 nitrogens are shown in orange, and cytosine amines are shown in blue. The location of bound hexammine ions is shown in green. The sequence of each variant is shown above the structure with boxes denoting the difference when compared to PM01. PM01 and PM02 differ in the relative orientation of their tandem G·U pairs, which moves the cation in the site. Both are examples of poor sites for localizing a hexammine. PM05 differs from PM01 by the orientation of a flanking pair, and it is an example of a good site with high occupancy and a high degree of cation localization (low Brel and high Anomrel, Table 1). Structures of the other tandem G·U pairs are contained in Supplemental Figure S1. b) Comparison of two representative single G·U pair-containing sequences. The sequence of each variant is shown above the structure with boxes denoting the difference when compared to PM13. PM14 contains a very good site, while PM16 has no hexammine in the pocket. Colors are the same as panel a, this figure. Structures of the other two single G·U pair-containing sequences are in Supplemental Figure S2.
Figure 4
Figure 4. General rules for cation binding in the major groove of G·U motifs
a) Schematic of the arrangement of major groove functional groups for PM01, PM05, and PM09. These three variants vary only in the orientation of flanking G-C pairs relative to the tandem G·U pairs. Blue circles are amine, red are carbonyls, and orange are purine N7 groups. In PM01, the two major groove amines (from cytosine) are placed away from the binding site due to the turn of the helix. In PM05 and PM09, the amines are placed in position to limit the mobility of the ion (shown in green). In addition, the location of carbonyls and N7 groups in the flanking sequences and close to the binding site make the ion more mobile as it attempts to satisfy multiple potential hydrogen binding partners. b) Schematic of the arrangement of major groove functional groups in the four single G·U pair containing variants. In single G·U pairs, the amines are best placed in the 3′ positions to withdraw them from the pocket.
Figure 5
Figure 5. Experimental electron density of RNAs phased using “at-home” and synchrotron radiation
a) Experimental electron density of the “high salt” crystal described in the text, obtained using diffraction data collected with an “at-home” X-ray source and SAD phasing from a cobalt (III) hexammine derivative. In red is an RNA model placed in the density. The asterisk denotes density of a cobalt (III) hexammine. This density is not ideal, but is clearly RNA. b) Experimental electron density of exactly the same RNA in panel a, but phased at a synchrotron using MAD data and iridium (III) hexammine. Again, the red shows placed RNA and the asterisks denote two bound iridium (III) hexammines. When compared to panel a, the density is greatly improved. c) Experimental electron density of the SRP RNA/M-domain complex using diffraction data collected with an “at-home” X-ray source and SAD phasing from a cobalt (III) hexammine derivative. In red is RNA placed in the density. Although this electron density was obtained using only SAD data from a rotating anode and cobalt (III) hexammine, the density rivals that obtained from synchrotron radiation and MAD phasing.
Figure 6
Figure 6. Flowchart of the phasing strategy integrated into existing RNA crystallography methods
Black boxes represent a standard methodology for producing diffracting RNA crystals and solving their structures, starting with design and purification of many different variants for use in crystallization screens. Our method integrates into this pathway (red boxes) without adding additional steps.
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References

    1. Adams PD, Gopal K, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, Pai RK, Read RJ, Romo TD, et al. Recent developments in the PHENIX software for automated crystallographic structure determination. J Synchrotron Radiat. 2004;11:53–55. - PubMed
    1. Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr. 2002;58:1948–1954. - PubMed
    1. Batey RT, Doudna JA. Structural and energetic analysis of metal ions essential to SRP signal recognition domain assembly. Biochemistry. 2002;41:11703–11710. - PubMed
    1. Batey RT, Gilbert SD, Montange RK. Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature. 2004;432:411–415. - PubMed
    1. Batey RT, Rambo RP, Lucast L, Rha B, Doudna JA. Crystal structure of the ribonucleoprotein core of the signal recognition particle. Science. 2000;287:1232–1239. - PubMed

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