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TitleA 'Build and Retrieve' methodology to simultaneously solve cryo-EM structures of membrane proteins.
Journal, issue, pagesNat Methods, Vol. 18, Issue 1, Page 69-75, Year 2021
Publish dateJan 6, 2021
AuthorsChih-Chia Su / Meinan Lyu / Christopher E Morgan / Jani Reddy Bolla / Carol V Robinson / Edward W Yu /
PubMed AbstractSingle-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein ...Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. We also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.
External linksNat Methods / PubMed:33408407 / PubMed Central
MethodsEM (single particle)
Resolution2.38 - 3.6 Å
Structure data

21897

6wti

EMDB-21897, PDB-6wti:
The Cryo-EM structure of the ubiquinol oxidase from Escherichia coli
Method: EM (single particle) / Resolution: 2.38 Å

21900

6wtz

EMDB-21900, PDB-6wtz:
Cryo-EM structure of E. Coli OmpF
Method: EM (single particle) / Resolution: 3.15 Å

21901

6wu0

EMDB-21901, PDB-6wu0:
CryoEM structure of Burkholderia pseudomallei hopanoid biosynthesis-associated RND transporter HpnN
Method: EM (single particle) / Resolution: 3.59 Å

21906

6wu6

EMDB-21906, PDB-6wu6:
succinate-coenzyme Q reductase
Method: EM (single particle) / Resolution: 3.6 Å

22528

7jz2

EMDB-22528, PDB-7jz2:
Succinate: quinone oxidoreductase SQR from E.coli K12
Method: EM (single particle) / Resolution: 2.5 Å

22529

7jz3

EMDB-22529, PDB-7jz3:
Osmoporin OmpC from E.coli K12
Method: EM (single particle) / Resolution: 2.56 Å

22530

7jz6

EMDB-22530, PDB-7jz6:
The Cryo-EM structure of the Catalase-peroxidase from Escherichia coli
Method: EM (single particle) / Resolution: 2.53 Å

22531

7jzh

EMDB-22531, PDB-7jzh:
The Cryo-EM structure of the Glutamate decarboxylase from Escherichia coli
Method: EM (single particle) / Resolution: 3.59 Å

Chemicals

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

ChemComp-HEO:
HEME O / Heme O

ChemComp-CU:
COPPER (II) ION / Copper

ChemComp-UQ8:
Ubiquinone-8

ChemComp-3PE:
1,2-Distearoyl-sn-glycerophosphoethanolamine / phospholipid*YM / Phosphatidylethanolamine

ChemComp-U9V:
pentadecyl(tetradecyl)peroxyanhydride

ChemComp-HOH:
WATER / Water

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER / Iron–sulfur cluster

ChemComp-SF4:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

ChemComp-F3S:
FE3-S4 CLUSTER / Iron–sulfur cluster

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM / Flavin adenine dinucleotide

ChemComp-NA:
Unknown entry

ChemComp-UQ2:
UBIQUINONE-2

Source
  • escherichia coli (E. coli)
  • Escherichia coli K-12 (bacteria)
  • escherichia coli (strain k12) (bacteria)
  • burkholderia pseudomallei mshr346 (bacteria)
  • escherichia coli 908573 (bacteria)
  • escherichia coli (strain se11) (bacteria)
KeywordsOXIDOREDUCTASE / ubiquinol oxidase cytochrome bo3 / membrane protein / outer membrane porin / omp / ompf / PROTEIN TRANSPORT / CryoEM structure / hopanoid transporter HpnN / ELECTRON TRANSPORT / Complex / succinate-coenzyme Q reductase / ELECTRON TRANSPORT/OXIDOREDUCTASE / Succinate: quinone oxidoreductase / SdhA / sdhB / SdhC / SdhD / ELECTRON TRANSPORT-OXIDOREDUCTASE complex / TRANSPORT PROTEIN / Osmoporin OmpC / catalase-peroxidase / LYASE / Glutamate decarboxylase beta

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