PDBsum entry 2npy

RNA

PDB id: 2npy

Contents

DNA/RNA

Ligands

NCO

S9L

Waters ×14

PDB id:

2npy

Name:

RNA

Title:

Crystal structure of a junctioned hairpin ribozyme incorporating 9atom linker and 2'-deoxy 2'-amino u at a-1

Structure:

5'-r( Up Cp Cp Cp (U2n)p Gp Up Cp Cp Ap Cp Cp G)-3'. Chain: a. Engineered: yes. Other_details: ribozyme substrate strand. 5'-r( Cp Gp Gp Up Gp Ap Gp Ap Ap Gp Gp G)-3'. Chain: b. Engineered: yes. Other_details: ribozyme domain linking strand. 5'-r( Gp Gp Cp Ap Gp Ap Gp Ap Ap Ap Cp Ap Cp Ap Cp Gp A)-

Source:

Synthetic: yes. Other_details: sequence occurs naturally in satellite RNA tobacco ringspot virus. Ringspot virus

Resolution:

2.65Å R-factor: 0.196 R-free: 0.223

Authors:

C.Macelrevey,J.Krucinska,J.E.Wedekind

Key ref:

C.MacElrevey et al. (2007). A posteriori design of crystal contacts to improve the X-ray diffraction properties of a small RNA enzyme. Acta Crystallogr D Biol Crystallogr, 63, 812-825. PubMed id: 17582172 DOI: 10.1107/S090744490702464X

Date:

30-Oct-06 Release date: 14-Aug-07

DNA/RNA chains

U-C-C-C-2AU-G-U-C-C-A-C-C-G 13 bases

C-G-G-U-G-A-G-A-A-G-G-G 12 bases

G-G-C-A-G-A-G-A-A-A-C-A-C-A-C-G-A 17 bases

U-C-G-U-G-G-U-A-C-A-U-U-A-C-C-U-G-C-C 19 bases

DOI no: 10.1107/S090744490702464X

Acta Crystallogr D Biol Crystallogr 63:812-825 (2007)

PubMed id: 17582172

A posteriori design of crystal contacts to improve the X-ray diffraction properties of a small RNA enzyme.

C.MacElrevey, R.C.Spitale, J.Krucinska, J.E.Wedekind.

ABSTRACT

The hairpin ribozyme is a small catalytic RNA comprising two helix-loop-helix domains linked by a four-way helical junction (4WJ). In its most basic form, each domain can be formed independently and reconstituted without a 4WJ to yield an active enzyme. The production of such minimal junctionless hairpin ribozymes is achievable by chemical synthesis, which has allowed structures to be determined for numerous nucleotide variants. However, abasic and other destabilizing core modifications hinder crystallization. This investigation describes the use of a dangling 5'-U to form an intermolecular U.U mismatch, as well as the use of synthetic linkers to tether the loop A and B domains, including (i) a three-carbon propyl linker (C3L) and (ii) a nine-atom triethylene glycol linker (S9L). Both linker constructs demonstrated similar enzymatic activity, but S9L constructs yielded crystals that diffracted to 2.65 A resolution or better. In contrast, C3L variants diffracted to 3.35 A and exhibited a 15 A expansion of the c axis. Crystal packing of the C3L construct showed a paucity of 6(1) contacts, which comprise numerous backbone to 2'-OH hydrogen bonds in junctionless and S9L complexes. Significantly, the crystal packing in minimal structures mimics stabilizing features observed in the 4WJ hairpin ribozyme structure. The results demonstrate how knowledge-based design can be used to improve diffraction and overcome otherwise destabilizing defects.

Selected figure(s)

Figure 2.
Figure 2 Ball-and-stick and electron-density maps of the engineered U·U mismatch at the top of helix 2 (H2). (a) The U-5·A14 base pair as observed in one of two molecules in the asymmetric unit of the 4WJ structure (PDB code 1m5k ). O atoms are colored red and N atoms are blue. Dashed gray lines indicate putative hydrogen bonds with corresponding distances. (b) The minimal junctionless ribozyme 64-mer structure (PDB code 1x9k ) with an orientation equivalent to that in (a). Two symmetry-related U-5 residues are separated by a 3 Å translation and are not base-paired. (c) The original syn-anti base-pairing conformation of U-5 modeled for the 2.05 Å resolution junctionless ribozyme (PDB code 1zfr ). Alternate conformations were modeled with equal occupancy and are colored green or cyan. A single set of hydrogen bonds is indicated; the symmetry-related hydrogen bonds and labels were omitted for clarity. A [A]-weighted simulated-annealing omit electron-density map calculated for the U-5 residue and nearby solvent molecules is shown contoured at 3.5 with coefficients mF[o] - DF[c]. (d) The revised 2.05 Å resolution minimal hairpin-ribozyme structure of this study depicting the new anti-anti or `Calcutta' model for U-5 fitted into an omit map as described in (c).

Figure 5.
Figure 5 Schematic surface and ball-and-stick diagrams illustrating the 6[1] packing interactions of hinged hairpin-ribozyme constructs. (a) Comparison of C3L (blue) and S9L (magenta) unit cells. The perspective represents the interaction of molecules about the 6[1] screw axis. (For twofold and 2[1] operations, refer to Fig. 6-a.) Superposition of C3L onto S9L initiates at the third molecule from the bottom (i.e. the `reference' molecule), represented in bold magenta and blue overlay. Symmetry operations used to generate the remaining molecules demonstrate the degeneration of the superposition as a result of the 15 Å elongated unit cell of the C3L structure. C3L molecules are shown in blue and green, with S9L structures shown in pink. The dashed box denotes the inset for (b) and (c). (b) Expanded view of the 6[1] packing scheme for the S9L structure. Atoms engaged in potential hydrogen bonds are depicted as white spheres. Helices are labeled as in Fig. 1-(c) and primes (') denote symmetry-related molecules. An additional H3'' helix that is packed in a blunt-ended base stack with H4 has been omitted for clarity. The asymmetric unit (H4) is colored magenta, with symmetry molecules in red (H3') or light pink (H4'). (c) The C3L 6[1] packing scheme as described in (b), but the asymmetric unit is colored blue and symmetry mates are coloured teal (H3') or green (H4'). White spheres identify equivalent atoms in the C3L structure that are engaged in hydrogen bonding in the S9L structure. (d)-(g) Detailed view of hydrogen-bond interactions with each of four H4 residues. The A31 and U31 residues are base-paired; A31 ends the linker strand, while U31 begins the S-turn strand. (h)-(k) Equivalent distances in the C3L structure demonstrate the loss of 6[1]-fold packing interactions.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2007, 63, 812-825) copyright 2007.

Figures were selected by an automated process.