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TitleMembrane protein isolation and structure determination in cell-derived membrane vesicles.
Journal, issue, pagesProc Natl Acad Sci U S A, Vol. 120, Issue 18, Page e2302325120, Year 2023
Publish dateMay 2, 2023
AuthorsXiao Tao / Chen Zhao / Roderick MacKinnon /
PubMed AbstractIntegral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of ...Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8 Å and 2.7 Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.
External linksProc Natl Acad Sci U S A / PubMed:37098056 / PubMed Central
MethodsEM (single particle)
Resolution2.7 - 3.8 Å
Structure data

EMDB-40038, PDB-8gh9:
Cryo-EM structure of hSlo1 in total membrane vesicles
Method: EM (single particle) / Resolution: 3.8 Å

EMDB-40044, PDB-8ghf:
cryo-EM structure of hSlo1 in plasma membrane vesicles
Method: EM (single particle) / Resolution: 2.7 Å

EMDB-40045, PDB-8ghg:
Cryo-EM structure of hSlo1 in digitonin, Ca2+-free and EDTA-free
Method: EM (single particle) / Resolution: 3.3 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-CA:
Unknown entry

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM / POPC

ChemComp-CLR:
CHOLESTEROL / Cholesterol

ChemComp-NA:
Unknown entry

Source
  • homo sapiens (human)
KeywordsTRANSPORT PROTEIN / Slo1 / BK channel / Ca2+- and voltage-activated K+ channel / ion channel / toal cell membrane vesicles / plasma membrane vesicles

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