Multi-crystal native-SAD phasing at 5 keV with a helium environment.

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Single-wavelength anomalous diffraction (SAD) using native sulfur or phosphorus (native-SAD) offers an alternative to heavy-atom derivatives for biomolecular structure determination.
• Native-SAD is especially valuable for membrane proteins due to difficulties in production and crystallization.
• Low-energy X-rays enhance native-SAD signals but are susceptible to increased background noise from air scattering and absorption.

Purpose of the Study:

• To develop and evaluate a helium path system to mitigate air scattering and absorption in low-energy native-SAD experiments.
• To demonstrate the feasibility of native-SAD phasing at 5 keV using a helium environment.
• To determine the structures of thaumatin and the membrane protein TehA using microcrystals and the developed helium path system.

Main Methods:

• Implementation of a helium path to reduce background noise and X-ray absorption at 5 keV.
• Collection of anomalous diffraction data from microcrystals of thaumatin and TehA within the helium path.
• Structure determination using data assembled from multiple microcrystals.

Main Results:

• The helium path effectively reduced background scattering and air absorption at 5 keV.
• Robust anomalous signals were obtained from assembled data of micro-sized crystals, despite weak individual signals.
• The structures of thaumatin (from 15 microcrystals) and TehA (from 18 microcrystals) were successfully determined.

Conclusions:

• A helium environment is crucial for successful native-SAD phasing at 5 keV.
• This approach enables structure determination of proteins, including challenging membrane proteins, from microcrystals.
• The developed helium path system significantly advances the application of native-SAD for biomolecular structure determination.