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Structure paper

TitleHigh-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM.
Journal, issue, pagesNat Commun, Vol. 10, Issue 1, Page 1032, Year 2019
Publish dateMar 4, 2019
AuthorsMark A Herzik / Mengyu Wu / Gabriel C Lander /
PubMed AbstractDetermining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While ...Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein.
External linksNat Commun / PubMed:30833564 / PubMed Central
MethodsEM (single particle)
Resolution2.8 - 6.0 Å
Structure data

0406

6nbb

EMDB-0406: Single-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase using 200 keV
PDB-6nbb: Horse liver alcohol dehydrogenase determined using single-particle cryo-EM at 200 keV
Method: EM (single particle) / Resolution: 2.9 Å

0407

6nbc

EMDB-0407: Single-particle cryo-EM reconstruction of human methemoglobin using 200 keV, state 1
PDB-6nbc: human methemoglobin state 1 determined using single-particle cryo-EM at 200 keV
Method: EM (single particle) / Resolution: 2.8 Å

0408

6nbd

EMDB-0408: Single-particle cryo-EM reconstruction of human methemoglobin using 200 keV, state 2
PDB-6nbd: Human methemoglobin state 2 determined using single-particle cryo-EM at 200 keV
Method: EM (single particle) / Resolution: 3.2 Å

EMDB-0409:
Single-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 using 200 keV
Method: EM (single particle) / Resolution: 6.0 Å

Chemicals

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM / Nicotinamide adenine dinucleotide

ChemComp-ZN:
Unknown entry

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

Source
  • equus caballus (horse)
  • homo sapiens (human)
  • Mus musculus (house mouse)
KeywordsOXIDOREDUCTASE / dehydrogenase / NADH-binding / homo-2-mer / OXYGEN TRANSPORT / heme-binding / hetero-4-mer / globin

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