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
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 microsco...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier BV
2022
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| Online Access: | https://hdl.handle.net/1721.1/146792 |
| _version_ | 1826194489203490816 |
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| author | Levitz, Talya S Brignole, Edward J Fong, Ivan Darrow, Michele C Drennan, Catherine L |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Levitz, Talya S Brignole, Edward J Fong, Ivan Darrow, Michele C Drennan, Catherine L |
| author_sort | Levitz, Talya S |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T09:56:52Z |
| format | Article |
| id | mit-1721.1/146792 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T09:56:52Z |
| publishDate | 2022 |
| publisher | Elsevier BV |
| record_format | dspace |
| spelling | mit-1721.1/1467922022-12-08T03:37:31Z 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, Talya S Brignole, Edward J Fong, Ivan Darrow, Michele C Drennan, Catherine L Massachusetts Institute of Technology. Department of Biology 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. 2022-12-07T18:49:30Z 2022-12-07T18:49:30Z 2022 2022-12-07T18:43:19Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/146792 Levitz, Talya S, Brignole, Edward J, Fong, Ivan, Darrow, Michele C and Drennan, Catherine L. 2022. "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." Journal of Structural Biology, 214 (1). en 10.1016/J.JSB.2021.107825 Journal of Structural Biology Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf Elsevier BV Elsevier |
| spellingShingle | Levitz, Talya S Brignole, Edward J Fong, Ivan Darrow, Michele C Drennan, Catherine L 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 |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| url | https://hdl.handle.net/1721.1/146792 |
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