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TitleEfficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.
Journal, issue, pagesProc Natl Acad Sci U S A, Vol. 120, Issue 31, Page e2302471120, Year 2023
Publish dateJul 24, 2023
AuthorsWooyoung Choi / Hao Wu / Klaus Yserentant / Bo Huang / Yifan Cheng /
PubMed AbstractCRISPR/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 ...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.
External linksProc Natl Acad Sci U S A / PubMed:37487103 / PubMed Central
MethodsEM (single particle)
Resolution1.98 - 2.3 Å
Structure data

29659

8g12

EMDB-29659, PDB-8g12:
CryoEM structure of nuclear GAPDH under 8h Oxidative Stress
Method: EM (single particle) / Resolution: 2.17 Å

29660

8g13

EMDB-29660, PDB-8g13:
CryoEM structure of cytosolic GAPDH under 8h Oxidative Stress
Method: EM (single particle) / Resolution: 2.3 Å

29661

8g14

EMDB-29661, PDB-8g14:
CryoEM structure of cytosolic GAPDH under 8h Oxidative Stress, class2
Method: EM (single particle) / Resolution: 2.3 Å

29662

8g15

EMDB-29662, PDB-8g15:
CryoEM structure of nuclear GAPDH under 24h Oxidative Stress
Method: EM (single particle) / Resolution: 2.07 Å

29663

8g16

EMDB-29663, PDB-8g16:
CryoEM structure of cytoplasmic GAPDH under 24h Oxidative Stress
Method: EM (single particle) / Resolution: 2.07 Å

29664

8g17

EMDB-29664, PDB-8g17:
CryoEM structure of wild-type GAPDH
Method: EM (single particle) / Resolution: 1.98 Å

Source
  • Homo (humans)
  • homo sapiens (human)
KeywordsTRANSFERASE / GAPDH / energy / cytosolic protein

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