A cryo-electron microscopic approach to elucidate protein structures from human brain microsomes

Abstract

We recently developed a “Build and Retrieve” cryo-electron microscopy (cryo-EM) methodology, which is capable of simultaneously producing near-atomic resolution cryo-EM maps for several individual proteins from a heterogeneous, multiprotein sample. Here we report the use of “Build and Retrieve” to define the composition of a raw human brain microsomal lysate. From this sample, we simultaneously identify and solve cryo-EM structures of five different brain enzymes whose functions affect neurotransmitter recycling, iron metabolism, glycolysis, axonal development, energy homeostasis, and retinoic acid biosynthesis. Interestingly, malfunction of these important proteins has been directly linked to several neurodegenerative disorders, such as Alzheimer’s, Huntington’s, and Parkinson’s diseases. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.

High-Resolution Structural Proteomics of Mitochondria Using the ‘Build and Retrieve’ Methodology

“…As with previous BaR applications ( 9 , 56 , 57 ), our methodology was able to identify and solve structures of proteins with relatively low (<10%) sample populations. By adding an additional 3D classification to our BaR pipeline, we also were able to distinguish and solve high-resolution structures of three very similar enzymes with 32 to 38% amino acid sequence similarity from the acyl-CoA dehydrogenase enzyme family.…”

Unveiling the Structural Proteome of an Alzheimer’s Disease Rat Brain Model

“…The BaR methodology offers a means to uncover structural details of diverse protein constituents within a raw biological sample, achieving near-atomic resolution 12 . The BaR method has previously been utilized to investigate protein complexes extracted from human brain microsomes 15 . However, the utilization of brain microsomes may present limitations in offering a native protein environment, intricate protein-protein interactions, a wider array of proteins, and a more accurate representation of cellular processes.…”

“…Here, we utilized Build and Retrieve (BaR) which is an iterative methodology that can carry out in silico purification and sorting of protein complexes within a large, diverse dataset from a heterogenous sample 12 . Deploying this method in image analysis has the capability to deconvolute images, facilitating the simultaneous generation of near-atomic resolution cryo-EM maps for individual proteins within a heterogeneous, multiprotein sample [12][13][14][15] .…”

Ex vivostructures from spinach leaves

“…For example, a bottom-up approach for structure determination of multiple enzyme complexes from the cellular milieu of the malaria-causing parasite Plasmodium falciparum has been described [17]. Moreover, a 'Build and Retrieve' method for determination of multiple protein complex structures from Escherichia coli lysate [18], and later also from human tissues [19][20][21][22].…”