8G15

CryoEM structure of nuclear GAPDH under 24h Oxidative Stress

Summary for 8G15

• Entry DOI: 10.2210/pdb8g15/pdb
• EMDB information: 29662
• Descriptor: Glyceraldehyde-3-phosphate dehydrogenase (1 entity in total)
• Functional Keywords: gapdh, energy, cytosolic protein, transferase
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 4
• Total formula weight: 143871.88

Authors

Choi, W.Y.,Wu, H.,Cheng, Y.F. (deposition date: 2023-02-01, release date: 2023-12-27)

Primary citation

Choi, W.,Wu, H.,Yserentant, K.,Huang, B.,Cheng, Y.
Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.
Proc.Natl.Acad.Sci.USA, 120:e2302471120-e2302471120, 2023

PubMed Abstract: CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.

PubMed: 37487103
DOI: 10.1073/pnas.2302471120

Experimental method

ELECTRON MICROSCOPY (2.07 Å)