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- EMDB-23773: Structure of the Neisseria gonorrhoeae ribonucleotide reductase i... -

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Basic information

Entry
Database: EMDB / ID: EMD-23773
TitleStructure of the Neisseria gonorrhoeae ribonucleotide reductase in the inactive state
Map data
Sample
  • Complex: Ribonucleotide reductase inactive complex
    • Protein or peptide: Ribonucleoside-diphosphate reductase subunit alphaRibonucleotide reductase
    • Protein or peptide: Ribonucleoside-diphosphate reductase subunit betaRibonucleotide reductase
  • Ligand: CYTIDINE-5'-DIPHOSPHATE
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: MU-OXO-DIIRON
  • Ligand: water
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
Ribonucleoside-diphosphate reductase / Ribonucleoside-diphosphate reductase
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsLevitz TS / Drennan CL / Brignole EJ
Funding support United States, 4 items
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, 2021-
Header (metadata) releaseJan 5, 2022-
Map releaseJan 5, 2022-
UpdateJan 5, 2022-
Current statusJan 5, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mdi
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mdi
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23773.png

Downloads & links

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Map

FileDownload / File: emd_23773.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.5998 Å
Density
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.27861944 - 0.47411168
Average (Standard dev.)3.4120498e-05 (±0.02169597)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 399.95 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.59981.59981.5998
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z399.950399.950399.950
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.2790.4740.000

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Supplemental data

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Sample components

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Entire : Ribonucleotide reductase inactive complex

EntireName: Ribonucleotide reductase inactive complex
Components
  • Complex: Ribonucleotide reductase inactive complex
    • Protein or peptide: Ribonucleoside-diphosphate reductase subunit alphaRibonucleotide reductase
    • Protein or peptide: Ribonucleoside-diphosphate reductase subunit betaRibonucleotide reductase
  • Ligand: CYTIDINE-5'-DIPHOSPHATE
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: MU-OXO-DIIRON
  • Ligand: water

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Supramolecule #1: Ribonucleotide reductase inactive complex

SupramoleculeName: Ribonucleotide reductase inactive complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Neisseria gonorrhoeae (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: T7 express
Molecular weightTheoretical: 528 KDa

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Macromolecule #1: Ribonucleoside-diphosphate reductase subunit alpha

MacromoleculeName: Ribonucleoside-diphosphate reductase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: ribonucleoside-diphosphate reductase
Source (natural)Organism: Neisseria gonorrhoeae (bacteria) / Strain: ATCC 700825 / FA 1090
Molecular weightTheoretical: 87.081344 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHEN LYFQMNTPTD LKVTKRDGRL EAIDLDKIHR VVTWAAEGLE NVSVSQVELK SHIQFYNGIR TDDIHETIIK AAADLISED TPDYQYLAAR LAIFHLRKIA YGEYEPPHLY NHVKKLTDAG KYDRHILEDY SREEFDELNA YIDHERDMSF S YAAVKQLE ...String:
MGHHHHHHEN LYFQMNTPTD LKVTKRDGRL EAIDLDKIHR VVTWAAEGLE NVSVSQVELK SHIQFYNGIR TDDIHETIIK AAADLISED TPDYQYLAAR LAIFHLRKIA YGEYEPPHLY NHVKKLTDAG KYDRHILEDY SREEFDELNA YIDHERDMSF S YAAVKQLE GKYLVQNRVT RQIYETPQFL YVLVAMCLFS KYPKEARLDY VKRFYDAVST FKVSLPTPIM SGVRTPTRQF SS CVLIECD DSLDSINATT SAIVKYVSQR AGIGINAGRI RGLDSEIRGG EARHTGCIPF FKMFQAAVKS CSQGGVRGGA ATL FYPLWH IEAESLLVLK NNRGVEDNRI RQLDYGVQIN RLLYTRLIKG GNITLFSPNE VSGLYEAFFA DQDEFERLYT KYEQ DPNIR KRIIPAADLF STLMQERAGT GRIYIQNVDH CNTHSPFDPR VAPVHQSNLC MEIALPTKPL DNINDPDGEI ALCTL SAFN LGALNSLDEL EGLADLTVRA LDALLDYQGY PVEAARTSTM DRRSLGIGVI NYAYYLAKNG VRYSDGSALG LTHRTF EAI QYYLLKASAN LAKEYGACTL FNQTVYSQGK LPIDTYKKDL DAVCGEPLHY DWESLRADIV KYGLRNSTLT ALMPSET SS QIANATNGIE PPRGLVTVKA SKDGILKQVV PEFETLKNAY ETLWQLPGNE GYLKLVGVMQ KFVDQAISAN TAYDPGKF E GNKVSMKQML KDLLTAYKYG VKTLYYHNTR DGADDTQTDI QDDGCAGGAC KI

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Macromolecule #2: Ribonucleoside-diphosphate reductase subunit beta

MacromoleculeName: Ribonucleoside-diphosphate reductase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO / EC number: ribonucleoside-diphosphate reductase
Source (natural)Organism: Neisseria gonorrhoeae (bacteria) / Strain: ATCC 700825 / FA 1090
Molecular weightTheoretical: 45.107785 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHEN LYFQMSYSTF PKTKNDALKE PMFFGQPVNV ARYDQQKYEV FEKLIEKQLS FFWRPEEIDV SRDRIDYANL PEHEKHIFI SNLKYQTLLD SIQGRSPNVA LLPLVSIPEL ETWVETWSFS ETIHSRSYTH IIRNIVNDPS VVFDDIVENE Y ITARAEDI ...String:
MGHHHHHHEN LYFQMSYSTF PKTKNDALKE PMFFGQPVNV ARYDQQKYEV FEKLIEKQLS FFWRPEEIDV SRDRIDYANL PEHEKHIFI SNLKYQTLLD SIQGRSPNVA LLPLVSIPEL ETWVETWSFS ETIHSRSYTH IIRNIVNDPS VVFDDIVENE Y ITARAEDI ACYYDDLIEY TQYYNLLGEG VHNVGGKPVT VSLRGLKKKL YLCLMCVNVL EAIRFYVSFA CSFAFAEREL ME GNAKIIK DIARDEALHL TGTQHMLNLM RSGVDDPEMA EIAAELQDEC FQLFKKAAEQ EKEWAAYLFK DGSMIGLNKE ILS QYVEYI TNLRMQAVGL PAGFEGANQN PIPWINAWLS SDNVQVAPQE VEISSYLIGQ IDSEVNTDDL GDFEL

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Macromolecule #3: CYTIDINE-5'-DIPHOSPHATE

MacromoleculeName: CYTIDINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: CDP
Molecular weightTheoretical: 403.176 Da
Chemical component information

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

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Macromolecule #4: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 8 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

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

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: MU-OXO-DIIRON

MacromoleculeName: MU-OXO-DIIRON / type: ligand / ID: 6 / Number of copies: 4 / Formula: FEO
Molecular weightTheoretical: 127.689 Da
Chemical component information

ChemComp-FEO:
MU-OXO-DIIRON

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 24 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6 mg/mL
BufferpH: 7.6
Component:
ConcentrationName
50.0 mMHEPES
15.0 mMMagnesium Chloride
1.0 mMEDTAEthylenediaminetetraacetic acid
3.06 percentGlycerol
VitrificationCryogen name: ETHANE
Details: Plunged using the chameleon (SPT labtech) Glow discharged at 12 mA for 250 s.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 92000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number grids imaged: 1 / Number real images: 620 / Average exposure time: 7.0 sec. / Average electron dose: 53.15 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 55161
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 50425
FSC plot (resolution estimation)

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