Strategic advances for cryo-EM structural studies of small (<100 kDa) GPCRs

Area of Science:

• Structural biology
• Biochemistry
• Pharmacology

Background:

• Membrane proteins, including G protein-coupled receptors (GPCRs), are crucial in cell signaling and represent a major drug target class.
• GPCRs comprise the largest membrane protein family, with approximately 35% of FDA-approved drugs targeting them.
• Cryo-electron microscopy (cryo-EM) has advanced GPCR structural studies, yet resolving small, flexible apo and inactive states remains difficult.

Purpose of the Study:

• To review challenges in visualizing apo and inactive GPCR states using cryo-electron microscopy (cryo-EM).
• To summarize fusion and target-binding strategies for determining GPCR structures.
• To discuss practical aspects of fiducial design and emerging AI approaches in GPCR structural biology.

Main Methods:

• Cryo-electron microscopy (cryo-EM) for structural determination.
• Fusion protein strategies to enhance stability and visualization.
• Target-binding approaches to stabilize specific GPCR conformations.
• Fiducial marker design for improved particle alignment.
• Artificial intelligence (AI) driven methods for image processing and structure prediction.

Main Results:

• Identification of key obstacles in cryo-EM for small, flexible apo/inactive GPCRs.
• Successful application of fusion and target-binding strategies to resolve challenging GPCR structures.
• Guidance on fiducial design for enhanced cryo-EM data quality.
• Highlighting the potential of AI to advance GPCR structural studies.

Conclusions:

• Overcoming cryo-EM limitations is crucial for understanding GPCRs in therapeutically relevant inactive states.
• Fusion and target-binding strategies offer viable solutions for structural determination of challenging GPCR complexes.
• Advancements in AI are poised to further revolutionize GPCR structural biology and drug discovery efforts.