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