Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

Abstract

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.

Keywords: C++; Phenix; Python; X-rays; automation; cctbx; cryo-EM; diffraction; macromolecular crystallography; neutrons.

Figure 1

Experimental methods used to determine macromolecular structures that are deposited in the PDB. The predominant method is X-ray diffraction, followed by nuclear magnetic resonance (NMR), cryo-EM and neutron diffraction.

Figure 2

Annual cryo-EM model depositions now outnumber X-ray model depositions in the resolution range 3.5–5 Å.

Figure 3

Since 2015, cryo-EM depositions have accounted for the majority of large macromolecular structures.

Figure 4

The structure-solution steps for X-ray/neutron crystallography and cryo-EM have nuances for each technique, but the overall workflow is similar. Color code: cryo-EM, gray; X-ray crystallography, green; neutron crystallography, red. As neutron diffraction experiments are typically performed with samples for which the structure is known, the phasing, density modification and model-building steps are not part of the workflow.

Figure 5

The primary tools for X-ray crystallography in Phenix.

Figure 6

The primary tools for cryo-EM in Phenix.

Figure 7

Cryo-EM maps deposited per year for different resolution ranges: better than 3 Å, 3–4 Å, 4–5 Å and worse than 5 Å.

Figure 8

Ramachandran φ, ψ plots for the pre-Pro case. (a) The reference distribution of 60 000 well determined pre-Pro residues, with contours that enclose the favored 98% of the data (thin green lines) and that exclude the outliers (thick green lines). (b) A pre-Pro Ramachandran plot in the Phenix GUI for a query structure, showing two labeled outliers in red. Note that pre-Pro is very different from a general case Ramachandran plot.

Figure 9

Screenshots of the cryo-EM Comprehensive validation tool and a Coot window. Clicking on the item in the table of cis/twisted peptides (highlighted in gray) recenters the Coot window on that peptide.

Figure 10

The videos on the Phenix tutorials YouTube channel cover the main Phenix programs, refinement strategies and lectures.