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Although the success of molecular-replacement techniques requires the solution of a six-dimensional problem, this is often subdivided into two three-dimensional problems. REMO09 is one of the programs which have adopted this approach. It has been revisited in the light of a new probabilistic approach which is able to directly derive conditional distribution functions without passing through a previous calculation of the joint probability distributions. The conditional distributions take into account various types of prior information: in the rotation step the prior information may concern a non-oriented model molecule alone or together with one or more located model molecules. The formulae thus obtained are used to derive figures of merit for recognizing the correct orientation in the rotation step and the correct location in the translation step. The phases obtained by this new version of REMO09 are used as a starting point for a pipeline which in its first step extends and refines the molecular-replacement phases, and in its second step creates the final electron-density map which is automatically interpreted by CAB, an automatic model-building program for proteins and DNA/RNA structures., (open access.)

The program Buccaneer, a well known fast and efficient automatic model-building program, is also a tool for phase refinement: indeed, input phases are used to calculate electron-density maps that are interpreted in terms of a molecular model, from which new phase estimates may be obtained. This specific property is shared by all other automatic model-building programs and allows their cyclic use, as is usually performed in other phase-refinement methods (for example electron-density modification techniques). Buccaneer has been included in a cyclic procedure, called CAB, aimed at increasing the rate of success of Buccaneer and the quality of the molecular models provided. CAB has been tested on 81 protein structures that were solved via molecular-replacement, anomalous dispersion and ab initio methods. The corresponding phases were submitted to a phase-refinement process that synergically combines current phase-refinement techniques and out-of-mainstream refinement methods [Burla et al. (2017), Acta Cryst. D73, 877-888]. The phases thus obtained were used as input for CAB. The experimental results were compared with those obtained by the sole use of Buccaneer: it is shown that CAB improves the Buccaneer results, both in completeness and in accuracy.

Ab initio and non-ab initio phasing methods are often unable to provide phases of sufficient quality to allow the molecular interpretation of the resulting electron-density maps. Phase extension and refinement is therefore a necessary step: its success or failure can make the difference between solution and nonsolution of the crystal structure. Today phase refinement is trusted to electron-density modification (EDM) techniques, and in practice to dual-space methods which try, via suitable constraints in direct and in reciprocal space, to generate higher quality electron-density maps. The most popular EDM approaches, denoted here as mainstream methods, are usually part of packages which assist crystallographers in all of the structure-solution steps from initial phasing to the point where the molecular model perfectly fits the known features of protein chemistry. Other phase-refinement approaches that are based on different sources of information, denoted here as out-of-mainstream methods, are not frequently employed. This paper aims to show that mainstream and out-of-mainstream methods may be combined and may lead to dramatic advances in the present state of the art. The statement is confirmed by experimental tests using molecular-replacement, SAD-MAD and ab initio techniques.