Guidelines for de novo phasing using multiple small-wedge data collection

Area of Science:

• Structural biology
• Crystallography
• Biophysics

Background:

• Intense micro-focus X-ray beamlines enable high-quality data collection from microcrystals of membrane proteins.
• Small-wedge synchrotron crystallography (SWSX) merges data from multiple microcrystals for structure determination.
• Previous studies lacked systematic investigation into dose dependence for SWSX.

Purpose of the Study:

• To investigate optimal X-ray dose conditions for experimental phasing using SWSX.
• To determine the impact of radiation dose on data accuracy and phase determination in SWSX.
• To identify the ideal dose range for de novo structure determination of membrane proteins via SWSX.

Main Methods:

• Utilized an automatic data collection system (ZOO) at SPring-8.
• Collected small-wedge (5-20°) datasets from multiple membrane protein microcrystals.
• Employed hierarchical clustering to group and merge homogeneous datasets with controlled doses.
• Analyzed dose dependence of data accuracy and phase determination using anomalous scattering signals.

Main Results:

• Phase determination via anomalous scattering signals became more challenging at higher X-ray doses.
• Merging homogeneous datasets with controlled doses mitigated issues like signal loss and radiation damage.
• Increased merging of datasets, even across a wider dose range, improved phase probability.
• A dose of 5 MGy was found to be optimal, balancing signal acquisition with minimized radiation damage.

Conclusions:

• Choosing a lower X-ray dose (< 10 MGy) is essential for de novo structure determination by SWSX.
• A dose of 5 MGy offers the best balance between data signal and minimizing radiation damage.
• Controlled dosing and dataset merging enhance data quality and phase determination in SWSX for membrane proteins.