Experimental Phasing

Why

One of the biggest challenges in crystallography is the phase problem. We measure amplitudes for structure factors, but cannot directly measure the phases. Unfortunately, these phases are necessary to generate the electron density of the molecules in the crystal.

Phases can be determined by experimental phasing, which relies on a few atoms in the macromolecule with known special properties, such as a large number of electrons and/or anomalous scattering. These properties are exploited to determine the location of the special atoms (substructure), and then knowledge of the substructure in one or more crystals is used to deduce phase information for the entire macromolecule. Thus, experimental phasing solves the phase problem by using the differences in amplitudes from different diffraction experiments to determine the structure factor phases. In modern crystallography, it is most common to use single or multiple anomalous diffraction experiments (SAD/MAD).

How

In Phenix, the primary program for performing experimental phasing is phenix.autosol. This is a comprehensive, automatic tool that performs experimental phasing with the MAD, MIR, SIR, or SAD methods. The program locates the substructure, estimates phases, performs density modification, identifies noncrystallographic symmetry, and builds and refines a preliminary model.

To run phenix.autosol, you need to provide diffraction data, typically one or more anomalous data sets, or a native data set plus one or more derivative data sets (if you are performing SIR/MIR phasing). You also need to provide the sequence of your macromolecule, and information about the anomalous scatterers and/or heavy atoms. The phenix.autosol results include a model file containing the anomalous scatterers, an MTZ file containing the experimentally determined phases, the optimized phases from density modification, and, if the maps are of sufficient quality, an atomic model.

How to use the phenix.autosol GUI: Click here

Phenix reference manual for phenix.autosol

Common issues

• Anomalous substructure not determined: There can be several reasons for this. Likely candidates are a lack of anomalous signal or an incorrect space group assignment. Use phenix.xtriage or phenix.anomalous_signal to estimate the resolution of the data for anomalous substructure determination. If this resolution is very low, it might not be possible to locate the substructure (note, the experimental sigmas are used in this calculation so it is important that these are well estimated). If there is uncertainty about the space group, it might be necessary to try the alternatives.
• An interpretable model isn't built: The most likely reason is that the electron density map is of insufficient quality to be automatically interpreted.
• Frequently asked questions about experimental phasing

Related programs

• phenix.plan_sad_experiment: This tool allows you to plan your SAD data collection strategy by estimating the anomalous signal and the probability of solving your structure with SAD based on the anomalously-scattering atoms, wavelength, size of your molecule, and overall I/sigI for your dataset.
• phenix.scale_and_merge: This tool scales any number of datasets together to obtain a merged, scaled anomalous dataset. It also produces two half-datasets of scaled anomalous data that can be used by phenix.anomalous_signal to accurately evaluate the anomalous signal in your data.
• phenix.anomalous_signal: This tool is like phenix.plan_sad_experiment except that it uses your measured data to give much more accurate estimates. Specifically, it uses your scaled data, two half-dataset files of your scaled data, the name of the anomalously-scattering atom, and the number of sites in the substructure in order to estimate the anomalous signal in your data and the probability of solving your structure by SAD. You can create the necessary scaled half-datasets using phenix.scale_and_merge.
• phenix.hyss: This hybrid substructure search program is used by phenix.autosol to locate the anomalous substructure or heavy atoms. It is possible to run this program separately (for anomalous data only) if you want to only find the substructure. In general, it is recommended to use phenix.autosol for this as it makes optimal use of phenix.hyss to locate sites.
• Phaser EP: Phaser can directly perform SAD phasing, given an initial anomalous substructure. Phases can be calculated for the substructure's given hand (enantiomer) or both hands. Phaser attempts to complete the substructure automatically. Note, Phaser can also combine information from a molecular replacement solution in the phase calculation process (in MR-SAD mode).
• phenix.emma: This Euclidian model matching program can compare different substructure solutions by superimposing sites taking into account crystallographic symmetry, possible origin shifts, and polar axes. This can be useful if comparing solutions from different programs.