Initiating heavy-atom-based phasing by multi-dimensional molecular replacement.

Area of Science:

• Structural Biology
• Crystallography
• Biophysics

Background:

• Solving the phase problem in macromolecular crystallography is crucial for obtaining electron-density maps.
• Heavy-atom substructure determination is often required but can fail with poorly diffracting crystals, common in membrane proteins.
• Existing methods like dual-space, direct, or Patterson-based approaches have limitations.

Purpose of the Study:

• To present a novel approach for heavy-atom site identification using a molecular-replacement parameter matrix (MRPM).
• To overcome limitations of current methods, particularly for challenging samples like membrane proteins.
• To enable accurate phasing for experimental structure determination.

Main Methods:

• Developed and applied a molecular-replacement parameter matrix (MRPM) approach.
• Performed an n-dimensional search testing various molecular-replacement parameters, including data sets and search models with different conformations.
• Scored results based on the ability to identify heavy-atom positions from anomalous difference Fourier maps.

Main Results:

• Successfully identified heavy-atom sites using the MRPM strategy.
• Applied the method to determine the structure of the copper-transporting P-type ATPase CopA, a membrane protein.
• Achieved successful heavy-atom substructure determination where previous methods failed.

Conclusions:

• The MRPM approach is effective for heavy-atom site identification, especially when dealing with poorly diffracting crystals.
• This method is well-suited for proteins with large conformational changes, allowing consideration of multiple search models.
• MRPM enables the identification of weak but correct molecular-replacement solutions, facilitating experimental phasing.