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.