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Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

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Published on: October 2, 2017

Data-collection strategy for challenging native SAD phasing.

Vincent Olieric¹, Tobias Weinert¹, Aaron D Finke¹

¹Swiss Light Source, Paul Scherrer Institut, Villigen PSI, Switzerland.

Acta Crystallographica. Section D, Structural Biology

|March 10, 2016

Summary

This summary is machine-generated.

Advancements in native single-wavelength anomalous diffraction (SAD) phasing enable macromolecular structure determination. Combining multiple datasets and low-dose X-ray collection overcomes challenges with difficult protein targets.

Keywords:

anomalous signal complex data-collection strategy membrane protein native SAD phasing

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Published on: April 23, 2021

Area of Science:

Structural biology
Biophysics
Crystallography

Background:

Native single-wavelength anomalous diffraction (SAD) phasing is crucial for determining macromolecular structures.
Improvements in data collection strategies are pushing the limits of this technique, especially for light elements.
Accurate measurements of anomalous differences are essential for successful phasing.

Purpose of the Study:

To describe the combined use of multiple data collection strategies for native SAD phasing.
To address challenges in phasing difficult targets like integral membrane proteins and large complexes.
To discuss optimal native SAD data collection strategies.

Main Methods:

Merging multiple datasets from multiple crystals or single crystals in multiple orientations.
Utilizing low X-ray doses to minimize radiation damage.
Applying these strategies to challenging protein targets such as diacylglycerol kinase (DgkA) and Cas9-RNA-DNA complex.

Main Results:

Successful native SAD phasing was achieved for challenging targets, including DgkA (1% Bijvoet ratio) and a large Cas9 complex in a low-symmetry space group.
High data multiplicity was obtained while minimizing radiation damage and systematic errors.
Accurate measurements of anomalous differences were ensured.

Conclusions:

The combined data collection strategies significantly enhance the capabilities of native SAD phasing.
This approach is effective for solving structures of previously challenging macromolecules.
Systematic measurements on tubulin complex inform optimal native SAD data collection strategies.