Molecular Replacement
Why
One of the biggest problems in crystallography is the phase problem,
hence the use of experimental phasing. An alternative approach, which is
increasingly popular, is the use of known structures to solve the
phase problem. This approach is called molecular replacement - a model,
very similar in structure to the one crystallized, is computationally
placed in the crystallographic unit cell. From this model, phases can be
calculated and used to start the process of interpreting the electron
density map.
How
In Phenix the primary program for performing molecular replacement is
Phaser. This requires experimental diffraction data, an atomic model(s),
the sequence of the molecule in the crystal and typically some estimate
of the number of molecules in the crystal. Phaser uses maximum
likelihood algorithms to determine the rotation of the model(s) with
respect to the unit cell, and then the translation of the rotated
model(s) within the unit cell. The output of this process, if
successful, is a PDB file containing the placed model(s) and an MTZ file
containing coefficients for the electron density made using the placed
model(s) and the observed experimental amplitudes. For an introduction
to molecular replacement in Phenix, click
here.
How to use the Phaser GUI in Phenix: Click
here
Common issues
- No solutions were found even with a good search model: There are
multiple causes, but one not uncommon reason is that a multiple
domain protein has undergone a conformational change. In this case
the molecular should be divided into the individual domains and the
molecular replacement performed with each domain as a separate search
model. An alternative cause, thankfully less common, is that the
crystallized molecule is not the intended one. For this reason it is
always prudent to use the "Search PDB Symmetry" tool in the Phenix
GUI to check cell dimensions and space group against the PDB.
A third possibility is that the space group is incorrect, for instance
if twinning increases the apparent symmetry of the diffraction pattern.
For this reason it is always good to run Xtriage in the "Data analysis"
section of the Phenix GUI.
- Frequently asked questions about molecular
replacement
Related programs
- phenix.MRage: This program uses Phaser to
perform highly automated molecular replacement. In its simplest form
it can take experimental diffraction data and a sequence to solve a
molecular replacement problem.
- phenix.sculptor: This program can be
used to prepare models for molecular replacement, by trimming loops
where there are expected to be insertions of deletions based on
sequence alignment. It can also trim side chains and modify other
properties of the model such as atomic displacement parameters.
- phenix.ensembler: This program can be
used to prepare ensembles for molecular replacement. An ensemble is a
set of structurally related models that have been superimposed with
respect to each other, typically using a conserved structural core.
Phenix.ensembler automates the process of superimposing models.
- phenix.morph_model: This program
can be used to improve an initial model after molecular replacement
by locally moving the structure to better fit the electron density
map. This is especially powerful in those cases where the molecular
replacement solution is structurally too different to provide phases
for map interpretation or automated model building.
- phenix.mr_rosetta: This program can
be used to improve an initial model after molecular replacement by
using the Rosetta program to modify the model such that it moves
closer to the true structure, while also improving the fit to
electron density map. This is especially powerful in those cases
where the molecular replacement solution is structurally too
different to provide phases for map interpretation or automated model
building.
Phenix reference manual for Phaser.