A case of structure determination using pseudosymmetry.

Area of Science:

• Structural Biology
• Crystallography
• Protein Structure Determination

Background:

• Group A Streptococcus utilizes cysteine protease Mac-1 (IdeS) to evade host immunity.
• Determining the structure of Mac-1 mutants is crucial for understanding its function and developing inhibitors.
• Crystallographic structure determination often faces challenges with unusual symmetries and phasing.

Purpose of the Study:

• To determine the crystal structure of the inactive C94A mutant of Mac-1.
• To investigate the utility of pseudosymmetry in facilitating structure determination when conventional methods fail.
• To elucidate the structural basis of Mac-1 function and immune evasion.

Main Methods:

• Crystallization of the Mac-1 C94A mutant in the monoclinic space group P2(1).
• Exploration of translational pseudosymmetry in the Patterson map, revealing pseudo-orthorhombic P2(1)2(1)2 symmetry.
• Single-wavelength anomalous diffraction (SAD) phasing using a subset of reflections obeying pseudosymmetry.
• Molecular replacement phasing for the full monoclinic dataset.

Main Results:

• The Mac-1 C94A mutant crystals exhibited monoclinic symmetry, but contained a subset of reflections obeying pseudo-orthorhombic symmetry.
• SAD phasing using the pseudo-orthorhombic subset successfully yielded interpretable electron density maps.
• The structure revealed six Mac-1 mutant molecules forming three dimers in the monoclinic unit cell.
• The geometric relationship between the pseudo-orthorhombic and monoclinic cells allowed direct generation of the full structure.

Conclusions:

• Pseudosymmetry can be effectively utilized as a phase-averaging method for anomalous diffraction structure determination.
• Solving a structure in a higher pseudosymmetry subcell, where NCS operators become crystallographic operators, is feasible.
• This approach successfully determined the Mac-1 mutant structure, overcoming challenges with conventional phasing methods.