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- PDB-7mdi: Structure of the Neisseria gonorrhoeae ribonucleotide reductase i... -

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Basic information

Entry
Database: PDB / ID: 7mdi
TitleStructure of the Neisseria gonorrhoeae ribonucleotide reductase in the inactive state
Components(Ribonucleoside-diphosphate reductase subunit ...) x 2
KeywordsOXIDOREDUCTASE / inactive complex / ribonucleotide reductase
Function / homology
Function and homology information


ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor / deoxyribonucleotide biosynthetic process / DNA replication / ATP binding
Similarity search - Function
Ribonucleotide reductase, class I , alpha subunit / Ribonucleotide reductase large subunit signature. / Ribonucleoside-diphosphate reductase large subunit / ATP-cone domain / ATP cone domain / ATP-cone domain profile. / Ribonucleotide reductase R1 subunit, N-terminal / Ribonucleotide reductase large subunit, N-terminal / Ribonucleotide reductase, all-alpha domain / Ribonucleotide reductase large subunit, C-terminal ...Ribonucleotide reductase, class I , alpha subunit / Ribonucleotide reductase large subunit signature. / Ribonucleoside-diphosphate reductase large subunit / ATP-cone domain / ATP cone domain / ATP-cone domain profile. / Ribonucleotide reductase R1 subunit, N-terminal / Ribonucleotide reductase large subunit, N-terminal / Ribonucleotide reductase, all-alpha domain / Ribonucleotide reductase large subunit, C-terminal / Ribonucleotide reductase, barrel domain / Ribonucleotide reductase small subunit, acitve site / Ribonucleotide reductase small subunit signature. / Ribonucleotide reductase small subunit / Ribonucleotide reductase small subunit family / Ribonucleotide reductase, small chain / Ribonucleotide reductase-like / Ferritin-like superfamily
Similarity search - Domain/homology
CYTIDINE-5'-DIPHOSPHATE / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / MU-OXO-DIIRON / Ribonucleoside-diphosphate reductase / Ribonucleoside-diphosphate reductase
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsLevitz, T.S. / Drennan, C.L. / Brignole, E.J.
Funding support United States, 4items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)2017246757 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM126982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM007287 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: J Struct Biol / Year: 2022
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.
Authors: Talya S Levitz / Edward J Brignole / Ivan Fong / Michele C Darrow / Catherine L Drennan /
Abstract: 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 ...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.
History
DepositionApr 5, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 5, 2022Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: Ribonucleoside-diphosphate reductase subunit alpha
E: Ribonucleoside-diphosphate reductase subunit beta
B: Ribonucleoside-diphosphate reductase subunit alpha
G: Ribonucleoside-diphosphate reductase subunit beta
D: Ribonucleoside-diphosphate reductase subunit alpha
F: Ribonucleoside-diphosphate reductase subunit beta
C: Ribonucleoside-diphosphate reductase subunit alpha
H: Ribonucleoside-diphosphate reductase subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)535,00432
Polymers528,7578
Non-polymers6,24724
Water43224
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: homology, high homology to the E. coli ribonucleotide reductase that has been shown to form a homologous structure using SAXS and analytical ultracentrifugation, microscopy, negative stain ...Evidence: homology, high homology to the E. coli ribonucleotide reductase that has been shown to form a homologous structure using SAXS and analytical ultracentrifugation, microscopy, negative stain electron microscopy (also single particle in vitro)
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Ribonucleoside-diphosphate reductase subunit ... , 2 types, 8 molecules ABDCEGFH

#1: Protein
Ribonucleoside-diphosphate reductase subunit alpha


Mass: 87081.344 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria) / Strain: ATCC 700825 / FA 1090 / Gene: NGO_0614 / Plasmid: pET30a / Production host: Escherichia coli (E. coli) / Strain (production host): T7 express
References: UniProt: Q5F8Z6, ribonucleoside-diphosphate reductase
#2: Protein
Ribonucleoside-diphosphate reductase subunit beta


Mass: 45107.785 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria) / Strain: ATCC 700825 / FA 1090 / Gene: NGO_0615 / Plasmid: pET30a / Production host: Escherichia coli (E. coli) / Strain (production host): T7 express
References: UniProt: Q5F8Z5, ribonucleoside-diphosphate reductase

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Non-polymers , 5 types, 48 molecules

#3: Chemical
ChemComp-CDP / CYTIDINE-5'-DIPHOSPHATE


Mass: 403.176 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H15N3O11P2
#4: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O12P3
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#6: Chemical
ChemComp-FEO / MU-OXO-DIIRON


Mass: 127.689 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe2O
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ribonucleotide reductase inactive complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.528 MDa / Experimental value: NO
Source (natural)Organism: Neisseria gonorrhoeae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: T7 express
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
215 mMMagnesium Chloride1
31 mMEDTA1
43.06 percentGlycerol1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE
Details: Plunged using the chameleon (SPT labtech) Glow discharged at 12 mA for 250 s

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 3100 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7 sec. / Electron dose: 53.15 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 620

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.3particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
10RELION3.0.8initial Euler assignment
11RELION3.0.8final Euler assignment
12RELION3.0.8classification
13RELION3.0.83D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 55161
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50425 / Symmetry type: POINT

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