A strategy for dissecting the architectures of native macromolecular assemblies

Area of Science:

• Structural biology
• Biochemistry
• Molecular cell biology

Background:

• Determining the structures of native macromolecular assemblies is difficult, particularly when they are present in low quantities within cells.
• Existing methods often struggle with the low abundance of endogenous complexes, hindering structural analysis.

Purpose of the Study:

• To develop a strategy for isolating and characterizing native macromolecular assemblies present at endogenous levels.
• To enable the structural modeling of low-abundance complexes using advanced biochemical techniques.

Main Methods:

• Utilized genetically engineered cell lines expressing Green Fluorescent Protein (GFP)-tagged proteins to isolate specific macromolecular complexes.
• Employed cross-linking mass spectrometry (XL-MS) to capture the spatial proximity of proteins within the complexes.
• Extracted distance restraints from XL-MS data to inform structural modeling.

Main Results:

• Successfully isolated macromolecular complexes at endogenous levels from GFP-tagged transgenic cell lines.
• Generated reliable distance restraints through cross-linking mass spectrometry.
• Developed computational models representing the molecular architectures of the isolated complexes.

Conclusions:

• The presented strategy effectively overcomes the challenge of low abundance for studying native macromolecular assemblies.
• Cross-linking mass spectrometry combined with GFP-tagging provides a powerful approach for structural modeling of cellular complexes.
• This method facilitates a deeper understanding of the organization and function of biological machinery in its native state.