Flowchart of the procedure used to solve the L1 ligase structure. An initial assembly of modeled RNA fragments (helices, loops) is used in combination with a native data set (F _obs) to obtain a starting molecular-replacement solution in Phaser (round_1.mtz and round_1.pdb). This solution is examined in Coot; all steric clashes are removed by manual editing and any nucleotide that does not reside in strong electron density in the σ_A-weighted 2F _o − F _c map is manually excised. The remaining model is then positionally refined using REFMAC (which can be performed within Coot) and any unoccupied density is modeled and refined in Coot. In addition, a new helical fragment (typically five base pairs of A-form helix) for a subsequent iteration of molecular replacement is generated. Using the edited and refined model as a fixed partial model, the next (ith) round of molecular replacement in Phaser and subsequent editing and refinement is carried out with the new helical fragment. This cycle is repeated until no more helical fragments can be added. At this point, at the Nth cycle, the best molecular-replacement solution (round_N.mtz and round_N.pdb) is used to generate a solvent-flattened electron-density map. The model itself (round_N.pdb) in the case of the L1 ligase was clearly flawed in that the positioning of the secondary-structural elements was not consistent with the primary sequence and it was clear that the ‘solution’ was one in which the secondary-structural fragments were generically positioned by Phaser without regard to the detailed sequence-based structure. For this reason, the model (round_N.pdb) was discarded and the calculated phase probability distributions were converted to Hendrickson–Lattmann coefficients within CCP4 and were then imported into CNS along with the native data set (F _obs) and treated as if they were experimentally determined MIR phases to be solvent-flattened. We employed solvent-flipping, using a solvent content of 50% predicted by the xtriage module of PHENIX, which simultaneously improved the quality of the electron-density map and helped to minimize model bias. The resulting map, pictured in Fig. 3 ▶, was then used to build the final model from scratch (i.e. without reference to the discarded molecular-replacement solution round_N.pdb), as if the map had been derived from experimental MIR phases. The resulting structure was then checked against a composite-OMIT map generated within CNS in which 10% of the model was omitted from each element of the composite and phases were regenerated from a standard simulated-annealing procedure with a starting temperature of 4000 K.

The final refined structure of the L1 ligase heterodimer (PDB code 2oiu). The final refined structure as described in detail in Robertson & Scott (2007 ▶). Each molecule varies from blue to red as one progresses in the 5′ to 3′ direction. The red/blue interface denotes the ligation-site junction.

The predicted secondary structure of the 71-nucleotide L1 ligase ribozyme. Sequences thought to be involved in canonical Watson–Crick (or wobble) base pairing or corresponding to the known GNRA tetraloop structure are colored in blue, green and yellow (a). Idealized models of these sequences were generated within Coot (b). The GAAA tetraloop was modeled using the previously determined minimal hammerhead RNA GUAA structure obtained from the PDB (299d).

The solvent-flattened ‘pseudo-MIR’ electron-density map. The electron-density map was generated in CNS using an initial phase probability distribution obtained from the iterative RNA-fragment molecular-replacement procedure outlined in Fig. 2 ▶ and described in the accompanying caption and in the main text.

The initial map. The σ_A-weighted 2F _o − F _c map, contoured at 2σ, produced from the initial four-fragment molecular-replacement solution. Additional helical density and some nucleotide base density is apparent in regions not modeled with the RNA fragments, indicating that the partial model possesses some degree of veracity and predictive phasing power. The color scheme is the same as that used in Fig. 1 ▶.