7mdi

Publication Abstract from PubMed

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 A resolution structure of Neisseria gonorrhoeae class Ia RNR in the inactive alpha4beta4 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 alpha4beta4 ring.

Effects 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.,Levitz TS, Brignole EJ, Fong I, Darrow MC, Drennan CL J Struct Biol. 2022 Mar;214(1):107825. doi: 10.1016/j.jsb.2021.107825. Epub 2021 , Dec 11. PMID:34906669^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.