Experimental Phasing

Why

One of the biggest problems 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. 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 powerful tool that goes through the steps of anomalous substructure location, phasing, phase improvement, and initial model building. To run phenix.autosol you will 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 will also need to provide the sequence of your macromolecule, and information about the anomalous scatterers and/or heavy atoms. The results from phenix.autosol will include a PDB containing the anomalous scatterers, an MTZ file containing the experimentally determined phases, and the optimized phases from density modification, and, if the maps are of sufficient quality a PDB file with an atomic model.

How to use the phenix.autosol GUI: Click here

Common issues

• Anomalous substructure not determined: there can be several reasons for this. Likely candidates are a lack of anomalous signal, or incorrect space group assignment. Use phenix.xtriage to estimate the resolution of the data for anomalous substructure determination. If this is very low resolution it might not be possible to locate the substructure (note that 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 (available in the GUI) allows you to plan your SAD data collection strategy by estimating the anomalous signal and the probability of solving your structure by SAD based on the anomalously-scattering atoms, the wavelength, the size of your molecule, and the overall I/sigI for your dataset.
• phenix.scale_and_merge: This tool allows you to scale any number of datasets together to obtain a merged, scaled anomalous dataset. It also produces two half-dataset scaled datasets 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. It uses your scaled data and two half-dataset scaled datasets from your data that can be created with phenix.scale_and_merge to give an estimate of the anomalous signal in your data and the probability of solving your structure by SAD, taking into account the number of sites in the substructure.
• phenix.hyss: This 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 be used directly to perform SAD phasing, given an initial anomalous substructure. Phases can be calculated for the given substructure hand or boths hands. Phaser will attempt to complete the substructure automatically. Note that Phaser can also combine information from a molecular replacement solution in the phase calculation process (in MR-SAD mode).
• phenix.emma: This program can be used to compare different substructure solution, by superimposing sites taking into account crystallographic symmetry, possible origin shifts, and polar axes. This can be useful if comparing solutions from different programs.

Phenix reference manual for phenix.autosol