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- EMDB-17360: Cryo-EM structure of the anaerobic ribonucleotide reductase from ... -

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Basic information

Entry
Database: EMDB / ID: EMD-17360
TitleCryo-EM structure of the anaerobic ribonucleotide reductase from Prevotella copri in its tetrameric state produced in the presence of dATP and CTP
Map dataMap post-processed using DeepEMhancer
Sample
  • Complex: Anaerobic ribonucleotide reductase from Prevotella copri in its tetrameric, dATP/CTP-bound state
    • Protein or peptide: Anaerobic ribonucleoside-triphosphate reductase
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsribonucleotide reductase glycyl radical enzyme allosteric regulation nucleotide biosynthesis / OXIDOREDUCTASE / BIOSYNTHETIC PROTEIN
Function / homologyRibonucleoside-triphosphate reductase, anaerobic / Anaerobic ribonucleoside-triphosphate reductase / ribonucleoside-triphosphate reductase (thioredoxin) activity / ATP-cone domain / ATP cone domain / ATP-cone domain profile. / DNA replication / ATP binding / Anaerobic ribonucleoside-triphosphate reductase
Function and homology information
Biological speciesPrevotella copri (bacteria) / Segatella copri (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsBanerjee I / Bimai O / Sjoberg BM / Logan DT
Funding support Sweden, United States, 4 items
OrganizationGrant numberCountry
Swedish Research Council2016-04855 Sweden
Swedish Research Council2019-01400 Sweden
CancerfondenCAN 20 1210 PjF Sweden
Wenner-Gren Foundation United States
CitationJournal: Elife / Year: 2024
Title: Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding.
Authors: Ornella Bimai / Ipsita Banerjee / Inna Rozman Grinberg / Ping Huang / Lucas Hultgren / Simon Ekström / Daniel Lundin / Britt-Marie Sjöberg / Derek T Logan /
Abstract: A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine ...A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from . The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.
History
DepositionMay 15, 2023-
Header (metadata) releaseSep 13, 2023-
Map releaseSep 13, 2023-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17360.png

Downloads & links

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Map

FileDownload / File: emd_17360.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap post-processed using DeepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.85 Å/pix.
x 448 pix.
= 379.187 Å		0.85 Å/pix.
x 448 pix.
= 379.187 Å		0.85 Å/pix.
x 448 pix.
= 379.187 Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8464 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.165
Minimum - Maximum-0.06729808 - 1.9304068
Average (Standard dev.)0.00081808836 (±0.020409497)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 379.1872 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17360_msk_1.map
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Additional map: Unsharpened map from non-uniform refinement in cryoSPARC

Fileemd_17360_additional_1.map
AnnotationUnsharpened map from non-uniform refinement in cryoSPARC
Projections & Slices
AxesZYX

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Half map: Half map A from non-uniform refinement in cryoSPARC

Fileemd_17360_half_map_1.map
AnnotationHalf map A from non-uniform refinement in cryoSPARC
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B from non-uniform refinement in cryoSPARC

Fileemd_17360_half_map_2.map
AnnotationHalf map B from non-uniform refinement in cryoSPARC
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Sample components

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Entire : Anaerobic ribonucleotide reductase from Prevotella copri in its t...

EntireName: Anaerobic ribonucleotide reductase from Prevotella copri in its tetrameric, dATP/CTP-bound state
Components
  • Complex: Anaerobic ribonucleotide reductase from Prevotella copri in its tetrameric, dATP/CTP-bound state
    • Protein or peptide: Anaerobic ribonucleoside-triphosphate reductase
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Anaerobic ribonucleotide reductase from Prevotella copri in its t...

SupramoleculeName: Anaerobic ribonucleotide reductase from Prevotella copri in its tetrameric, dATP/CTP-bound state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Prevotella copri (bacteria)
Molecular weightTheoretical: 336.8 KDa

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Macromolecule #1: Anaerobic ribonucleoside-triphosphate reductase

MacromoleculeName: Anaerobic ribonucleoside-triphosphate reductase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Segatella copri (bacteria)
Molecular weightTheoretical: 84.636055 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPGSMIQTVV KRDGRIVGFN EQKIMAAIRK AMLHTDKGED TTLIEQITDH ISYRGKSQMS VEAIQDAIEM ELMKSARKDV AQKYIAYRN QRNIARKAKT RDVFMSIVNA KNNDITRENA NMNADTPAGM MMKFASETTK PFVDDYLLSE DVRDAVMHNY I HIHDKDYY ...String:
GPGSMIQTVV KRDGRIVGFN EQKIMAAIRK AMLHTDKGED TTLIEQITDH ISYRGKSQMS VEAIQDAIEM ELMKSARKDV AQKYIAYRN QRNIARKAKT RDVFMSIVNA KNNDITRENA NMNADTPAGM MMKFASETTK PFVDDYLLSE DVRDAVMHNY I HIHDKDYY PTKSLTCVQH PLDVILNHGF TAGHGSSRPA KRIETAAVLA CISLETCQNE MHGGQAIPAF DFYLAPYVRM SY QEEVKNL EKLTGEDLSN LYDAPIDDYI EKPLDGLQGR ERLEQHAINK TVNRVHQAME AFIHNMNTIH SRGGNQVVFS SIN YGTDTS AEGRCIMREI LQSTYQGVGN GETAIFPIQI WKKKRGVNYL PEDRNYDLYK LACKVTARRF FPNFLNLDAT FNQN EKWRA DDPERYKWEI ATMGCRTRVF EDRWGEKTSI ARGNLSFSTI NIVKLAIECM GIENEKQRID MFFAKLDNIL DITAK QLDE RFQFQKTAMA KQFPLLMKYL WVGAENLKPE ETIESVINHG TLGIGFIGLA ECLVALIGKH HGESEKAQEL GLKIIT YMR DRANEFSEQY HHNYSILATP AEGLSGKFTK KDRKQFGVIP GVTDRDYYTN SNHVPVYYKC TALKKAQIEA PYHDLTR GG HIFYVEIDGD ATHNPSVIES VVDMMDKYNM GYGSVNHNRN RCLDCGYENA DAHLEVCPKC GSHHIDKLQR ITGYLVGT T DRWNSGKLAE LHDRVTHIGG EK

UniProtKB: Anaerobic ribonucleoside-triphosphate reductase

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

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 12 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES-NaOHHEPES buffer
100.0 mMKClpotassium chloride
0.5 mMTCEPtris(2-carboxyethyl)phosphine
10.0 mMMgCl2magnesium chloride
0.5 mMdATPdeoxyadenosine triphosphate
0.5 mMCTPcytosine triphosphate
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 1, 5s blot time.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 11780 / Average exposure time: 2.0 sec. / Average electron dose: 38.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5762760
Startup modelType of model: OTHER / Details: ab initio model from cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 1105348
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8p27


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsManual fitting was done using Coot and automatic real space refinement used phenix.refine
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: correlation coefficient
Output model

PDB-8p2c:
Cryo-EM structure of the anaerobic ribonucleotide reductase from Prevotella copri in its tetrameric state produced in the presence of dATP and CTP

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