Exploiting large non-isomorphous differences for phase determination of a G-segment invertase-DNA complex

Area of Science:

• Structural Biology
• Biochemistry
• Crystallography

Background:

• G-segment invertase is crucial for DNA recombination.
• Determining the structure of enzyme-DNA complexes is vital for understanding their mechanisms.
• Previous attempts to resolve the G-segment invertase-DNA complex were hindered by challenging crystal properties.

Purpose of the Study:

• To determine the high-resolution structure of the G-segment invertase in complex with its DNA substrate.
• To overcome challenges posed by non-isomorphism and anisotropy in crystal diffraction data.
• To elucidate the conformation of the DNA substrate when bound to G-segment invertase.

Main Methods:

• Crystallization of the G-segment invertase-DNA complex.
• X-ray diffraction data collection from challenging crystals (88% solvent content, 5.0 Å resolution).
• Development and application of a cross-crystal averaging procedure incorporating non-isomorphous data and prior molecular boundary information.

Main Results:

• Obtained high-quality experimental phases despite significant crystallographic challenges.
• The structure revealed a unique conformation of the 37-base-pair asymmetric DNA duplex substrate.
• The bound DNA conformation differs significantly from substrates observed in other serine recombinase-DNA complexes.

Conclusions:

• The developed cross-crystal averaging method is effective for structure determination from difficult crystallographic datasets.
• The unique DNA substrate conformation provides new insights into the mechanism of G-segment invertase-mediated DNA recombination.
• This structural information can inform the design of novel genetic tools and therapeutic strategies targeting DNA recombination.