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TitleEffects of chameleon dispense-to-plunge speed on particle concentration, complex formation, and final resolution: A case study using the Neisseria gonorrhoeae ribonucleotide reductase inactive complex.
Journal, issue, pagesJ Struct Biol, Vol. 214, Issue 1, Page 107825, Year 2022
Publish dateDec 11, 2021
AuthorsTalya S Levitz / Edward J Brignole / Ivan Fong / Michele C Darrow / Catherine L Drennan /
PubMed AbstractRibonucleotide reductase (RNR) is an essential enzyme that converts ribonucleotides to deoxyribonucleotides and is a promising antibiotic target, but few RNRs have been structurally characterized. We ...Ribonucleotide reductase (RNR) is an essential enzyme that converts ribonucleotides to deoxyribonucleotides and is a promising antibiotic target, but few RNRs have been structurally characterized. We present the use of the chameleon, a commercially-available piezoelectric cryogenic electron microscopy plunger, to address complex denaturation in the Neisseria gonorrhoeae class Ia RNR. Here, we characterize the extent of denaturation of the ring-shaped complex following grid preparation using a traditional plunger and using a chameleon with varying dispense-to-plunge times. We also characterize how dispense-to-plunge time influences the amount of protein sample required for grid preparation and preferred orientation of the sample. We demonstrate that the fastest dispense-to-plunge time of 54 ms is sufficient for generation of a data set that produces a high quality structure, and that a traditional plunging technique or slow chameleon dispense-to-plunge times generate data sets limited in resolution by complex denaturation. The 4.3 Å resolution structure of Neisseria gonorrhoeae class Ia RNR in the inactive α4β4 oligomeric state solved using the chameleon with a fast dispense-to-plunge time yields molecular information regarding similarities and differences to the well studied Escherichia coli class Ia RNR α4β4 ring.
External linksJ Struct Biol / PubMed:34906669 / PubMed Central
MethodsEM (single particle)
Resolution4.3 Å
Structure data

EMDB-23773, PDB-7mdi:
Structure of the Neisseria gonorrhoeae ribonucleotide reductase in the inactive state
Method: EM (single particle) / Resolution: 4.3 Å

Chemicals

ChemComp-CDP:
CYTIDINE-5'-DIPHOSPHATE / Cytidine diphosphate

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate

ChemComp-MG:
Unknown entry

ChemComp-FEO:
MU-OXO-DIIRON

ChemComp-HOH:
WATER / Water

Source
  • neisseria gonorrhoeae (bacteria)
KeywordsOXIDOREDUCTASE / inactive complex / ribonucleotide reductase

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