A 'Build and Retrieve' methodology to simultaneously solve cryo-EM structures of membrane proteins

Area of Science:

• Structural Biology
• Biochemistry
• Molecular Biology

Background:

• Single-particle cryo-electron microscopy (cryo-EM) is crucial for structural biology.
• Obtaining pure, homogeneous membrane protein samples for cryo-EM is a significant challenge.
• This limitation hinders the structural determination of many important membrane proteins.

Purpose of the Study:

• To develop a novel method for determining cryo-EM structures from heterogeneous and impure membrane protein samples.
• To enable the identification and structural analysis of less abundant and smaller membrane proteins.
• To demonstrate the applicability of the method to crude biological samples.

Main Methods:

• Development of the Build and Retrieve (BaR) bottom-up iterative method.
• Application of BaR to heterogeneous, impure membrane protein samples.
• Analysis of Escherichia coli K12 crude membrane and raw lysate samples.

Main Results:

• Successful identification and determination of cryo-EM structures for various inner and outer membrane proteins and complexes.
• Elucidation of structural information from complex biological mixtures like crude membranes and lysates.
• High-resolution structures were obtained for small (<100 kDa) and low-abundance (<10%) unidentified membrane proteins within a single heterogeneous sample.

Conclusions:

• The BaR methodology significantly advances the structural determination of membrane proteins using cryo-EM.
• This approach overcomes the challenge of sample purity and heterogeneity.
• Cryo-EM, coupled with BaR, shows great potential for systems structural proteomics and uncovering the structures of previously uncharacterized membrane proteins.