Ab initio phase determination for X-ray diffraction data from crystals of a native protein.

Area of Science:

• Structural Biology
• Biophysics
• Computational Chemistry

Background:

• Macromolecular structure determination is crucial for understanding biological function.
• Traditional methods like molecular replacement have limitations for large or novel protein structures.
• Accurate phase determination is essential for resolving electron density maps in X-ray crystallography.

Purpose of the Study:

• To apply an efficient maximum entropy algorithm for determining X-ray diffraction phases.
• To test the feasibility of this method for a large protein, recombinant bovine chymosin.
• To demonstrate the potential of maximum entropy methods (MEM) in macromolecular crystallography.

Main Methods:

• Utilized an efficient algorithm for positive electron-density distribution determination.
• Employed a systematic procedure for selecting centric reflection phases using map entropy as a figure of merit.
• Iteratively added acentric and centric reflections, optimizing phase choices based on total map entropy.

Main Results:

• Successfully determined X-ray diffraction phases for recombinant bovine chymosin (323 amino acids).
• A low-resolution map showed remarkable agreement with the refined structure.
• 141 out of 159 centric reflections (89%) and a median absolute phase difference of 32 degrees for 1811 acentric reflections were obtained.
• The method was applied to a molecule significantly larger than those previously solved without specialized phasing techniques.

Conclusions:

• Maximum entropy methods offer a powerful approach for macromolecular phase determination.
• This study highlights the potential of MEM for structures determined without isomorphous replacement, molecular replacement, or anomalous dispersion.
• The successful phasing of recombinant bovine chymosin demonstrates the scalability and effectiveness of MEM for complex biological molecules.