Cryo-EM and artificial intelligence visualize endogenous protein community members

Area of Science:

• Structural biology
• Molecular systems biology
• Cellular biochemistry

Background:

• Cellular functions rely on large protein assemblies organized into communities called metabolons.
• Metabolons are involved in diverse metabolic pathways, including protein, fatty acid, and thioester synthesis.
• The heterogeneity of metabolons presents significant analytical challenges.

Purpose of the Study:

• To simultaneously characterize the in-situ architectures of key metabolon-embedded complexes: 60S pre-ribosome, fatty acid synthase, and pyruvate/oxoglutarate dehydrogenase E2 cores.
• To develop and apply an integrated approach combining cryo-electron microscopy (cryo-EM) with AI-driven modeling for analyzing native cellular structures.
• To explore the potential functional implications of observed conformational variations in cellular components.

Main Methods:

• Utilized cryo-electron microscopy (cryo-EM) to obtain 3D reconstructions of cellular fractions at 3.84-4.52 Å resolution.
• Employed artificial intelligence-based atomic modeling and de novo sequence identification for structural analysis.
• Collected fewer than 3,000 micrographs from a single cellular fraction for efficient data acquisition.

Main Results:

• Successfully resolved the de novo architectures of metabolon-embedded 60S pre-ribosome, fatty acid synthase, and pyruvate/oxoglutarate dehydrogenase E2 cores.
• Identified discernible polypeptide hydrogen bonding patterns at the achieved resolution, enabling detailed structural insights.
• Observed that resident molecular components are similar to purified eukaryotic counterparts but display significant conformational variations.

Conclusions:

• The study presents an integrated, machine learning-boosted tool for analyzing native cellular extracts.
• This approach facilitates structural systems biology by enabling high-resolution cryo-EM characterization of complex cellular architectures.
• The findings highlight the conformational plasticity of cellular components within metabolons, suggesting functional relevance.