Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.

phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used separately or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a critical component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods.

This article describes the implementation of real-space refinement in thephenix.real_space_refineprogram from thePHENIXsuite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refinement of atomic models against low-resolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry,phenix.real_space_refinemakes use of extra information such as secondary-structure and rotamer-specific restraints, as well as restraints or constraints on internal molecular symmetry. The re-refinement of 385 cryo-EM-derived models available in the Protein Data Bank at resolutions of 6 Å or better shows significant improvement of the models and of the fit of these models to the target maps.

Recent advances in the field of electron cryomicroscopy (cryo-EM) have resulted in a rapidly increasing number of atomic models of biomacromolecules that have been solved using this technique and deposited in the Protein Data Bank and the Electron Microscopy Data Bank. Similar to macromolecular crystallography, validation tools for these models and maps are required. While some of these validation tools may be borrowed from crystallography, new methods specifically designed for cryo-EM validation are required. Here, new computational methods and tools implemented inPHENIXare discussed, includingd₉₉to estimate resolution,phenix.auto_sharpento improve maps andphenix.mtriageto analyze cryo-EM maps. It is suggested that cryo-EM half-maps and masks should be deposited to facilitate the evaluation and validation of cryo-EM-derived atomic models and maps. The application of these tools to deposited cryo-EM atomic models and maps is also presented.

A crystallographic macromolecular model is typically characterized by a list of quality criteria, such as R factors, deviations from ideal stereochemistry and average B factors, which are usually provided as tables in publications or in structural databases. In order to facilitate a quick model-quality evaluation, a graphical representation is proposed. Each key parameter such as R factor or bond-length deviation from ;ideal values' is shown graphically as a point on a ;ruler'. These rulers are plotted as a set of lines with the same origin, forming a hub and spokes. Different parts of the rulers are coloured differently to reflect the frequency (red for a low frequency, blue for a high frequency) with which the corresponding values are observed in a reference set of structures determined previously. The points for a given model marked on these lines are connected to form a polygon. A polygon that is strongly compressed or dilated along some axes reveals unusually low or high values of the corresponding characteristics. Polygon vertices in ;red zones' indicate parameters which lie outside typical values.

Refinement of atomic models is a necessary step in solving the macromolecular structure by X-ray diffraction methods. Nowadays, high automation and well-developed interfaces give a possibility to use the most popular refinement programs as black boxes. Nevertheless, working with complex objects requires an understanding of the internal structure and principles of operation of these programs and critical assessment of the results of refinement. In this review, we discuss the basic principles of the organization of refinement programs and the history of their improvement and development, as the studied objects became more and more complicated. The discussions are kept at the level of basic mathematic knowledge avoiding unnecessary formalism and too detailed expressions.