Structures from anomalous diffraction of native biological macromolecules.

Area of Science:

• Structural Biology
• Crystallography
• Biophysics

Background:

• Solving the crystallographic phase problem is crucial for de novo structure determination of novel biological macromolecules.
• Conventional methods often rely on incorporating heavy atoms, such as using selenomethionyl proteins for multi-wavelength anomalous diffraction (MAD) or single-wavelength anomalous diffraction (SAD) experiments.

Purpose of the Study:

• To develop and validate a routine method for de novo crystal structure determination using intrinsic anomalous scattering from native macromolecules.
• To provide an alternative to selenomethionyl SAD experiments that bypasses the need for heavy-atom incorporation.

Main Methods:

• Developed robust procedures to enhance signal-to-noise ratio from native anomalous scattering.
• Employed a multicrystal SAD approach, combining data from multiple crystals (5 to 13) at lower X-ray energies.
• Collected data at lower-than-usual X-ray energies to amplify anomalous scattering signals.

Main Results:

• Successfully determined native protein structures at modest resolutions (2.3 to 2.8 angstroms).
• Applied the method to proteins of varying sizes (127 to 1148 residues) and sulfur atom counts (3 to 28).
• Demonstrated routine structure determination without the need for heavy-atom incorporation.

Conclusions:

• The multicrystal SAD method using intrinsic anomalous scattering is a viable and attractive alternative for de novo structure determination.
• This technique simplifies the process by eliminating the requirement for heavy-atom derivatization, making structural biology more accessible.
• The findings pave the way for more efficient structure determination of novel biological macromolecules.