Microcrystallography of Protein Crystals and In Cellulo Diffraction

Area of Science:

• Structural Biology
• Biophysics
• X-ray Crystallography

Background:

• Analyzing protein microcrystals (<10 µm) by X-ray crystallography was challenging.
• Advancements in microfocus beamlines enable routine analysis of small crystals.

Purpose of the Study:

• To present and compare two workflows for protein microcrystal structure determination.
• Focus on crystals grown in vivo (within cells).

Main Methods:

• Two workflows: 1) In vitro purification (sonication, centrifugation) of microcrystals. 2) In cellulo analysis using flow cytometry.
• Optional heavy atom soaks for phasing.
• Sample preparation: micromesh support, flash cooling.
• Serial diffraction experiments at microfocus synchrotron beamlines.

Main Results:

• Both workflows yield datasets suitable for phasing, model building, and refinement.
• In cellulo analysis proved more efficient than purified crystal analysis.
• Structure determination of Bombyx mori cypovirus 1 (BmCPV1) polyhedrin achieved in ~8 days.

Conclusions:

• Established workflows facilitate protein microcrystal structure determination.
• In cellulo analysis offers an efficient approach for in vivo grown crystals.
• Accelerated structure determination is possible for challenging samples.