Molecular Replacement

Why

One of the biggest challenges in crystallography is the phase problem, which can be addressed with experimental phasing methods. A popular alternative approach is molecular replacement (MR), which uses known structures to solve the phase problem. When a structurally similar model (e.g., a homologue) is available, this model is computationally placed in the unit cell containing the unknown structure. An initial electron density map is then calculated with the phases from the homologue and the observed structure factors.

The success of MR calculations depends on how much signal can be extracted from the data using the particular model, which in turn depends on a combination of the model quality and completeness, data resolution, and number of diffraction observations. For crystals of medium-sized proteins diffracting to moderate resolution, for example, the sequence identity between the molecule and homologue should be >25-30% and the r.m.s. deviation between C⍺ atoms should be <2.0 Å.

How

In Phenix, the primary program for performing molecular replacement is Phaser. This requires experimental diffraction data, 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 model 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

Phenix reference manual for Phaser.

Common issues

• No solutions were found even with a good search model: There are multiple causes, but a common one is that a multiple domain protein has undergone a conformational change. In this case, you should split the structure and perform molecular replacement on the individual domains using separate search models. 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 the cell dimensions and space group against the PDB. A third possibility is that the space group is incorrect (e.g., if twinning increases the apparent symmetry of the diffraction pattern), thus 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 uses experimental diffraction data and a sequence to solve a molecular replacement problem.
• phenix.sculptor: This program can improve models for molecular replacement by trimming off parts of the model that are unlikely to be preserved in the target structure.
• phenix.ensembler: This program prepares 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 improve an initial model after molecular replacement by locally moving the structure to better fit the electron density map. This is especially powerful when the molecular replacement solution is structurally too different to provide phases for map interpretation or automated model building.
• phenix.mr_rosetta: This program can 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 when the molecular replacement solution is structurally too different to provide phases for map interpretation or automated model building.