Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

Area of Science:

• Structural biology
• Biophysics
• Crystallography

Background:

• X-ray crystallography is crucial for determining biological macromolecule structures.
• Obtaining large, well-diffracting crystals has historically been a significant challenge.
• Advancements in serial crystallography and multi-crystal methods reduce the need for large crystals.

Purpose of the Study:

• To present an automated protocol for collecting diffraction data from micro-crystals.
• To demonstrate the utility of this method for structure determination when large crystals are unavailable.
• To showcase the application of MeshAndCollect for solving the crystal structure of Cerulean Cyan Fluorescent Protein.

Main Methods:

• Utilized the automated MeshAndCollect protocol for micro-crystal analysis.
• Identified positions of multiple small crystals on a single sample holder.
• Directed serial data collection of partial diffraction datasets from micro-crystals.
• Merged diffraction data for subsequent structure determination.

Main Results:

• Successfully applied MeshAndCollect to micro-crystals, including weakly diffracting ones.
• Demonstrated the protocol's effectiveness in overcoming crystal size limitations.
• Solved the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean using this technique.

Conclusions:

• The MeshAndCollect protocol facilitates X-ray crystallography structure determination from micro-crystals.
• This method significantly reduces the bottleneck associated with crystal growth.
• Enables structural analysis of biological macromolecules previously limited by crystal availability.