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- EMDB-12985: Low resolution reconstruction of the dATP-inhibited complex of th... -

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Basic information

Entry
Database: EMDB / ID: EMD-12985
TitleLow resolution reconstruction of the dATP-inhibited complex of the ribonucleotide reductase NrdA and NrdB proteins from Leeuwenhoekiella blandensis
Map data
Sample
  • Complex: dATP-inhibited complex of NrdA and NrdB subunits from Leeuwenhoekiella blandensis
    • Protein or peptide: Ribonucleotide reductase catalytic subunit from Leeuwenhoekiella blandensis
    • Protein or peptide: Ribonucleotide reductase radical generating subunit from Leeuwenhoekiella blandensis
Biological speciesLeeuwenhoekiella blandensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsBanerjee I / Rozman Grinberg I / Sjoberg BM / Logan DT
Funding support Sweden, 4 items
OrganizationGrant numberCountry
Swedish Research Council2016-04855 Sweden
Swedish Research Council2019-01400 Sweden
Wenner-Gren Foundation Sweden
Cancerfonden2018/820 Sweden
CitationJournal: Front Mol Biosci / Year: 2021
Title: Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From by SAXS and Cryo-Electron Microscopy.
Authors: Mahmudul Hasan / Ipsita Banerjee / Inna Rozman Grinberg / Britt-Marie Sjöberg / Derek T Logan /
Abstract: The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall ...The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall activity is downregulated by the binding of dATP to a small domain known as the ATP-cone often found at the N-terminus of RNR subunits, causing oligomerization that prevents formation of a necessary αβ complex between the catalytic (α) and radical generating (β) subunits. In some relatively rare organisms with RNRs of the subclass NrdAi, the ATP-cone is found at the N-terminus of the β subunit rather than more commonly the α subunit. Binding of dATP to the ATP-cone in β results in formation of an unusual β tetramer. However, the structural basis for how the formation of the active complex is hindered by such oligomerization has not been studied. Here we analyse the low-resolution three-dimensional structures of the separate subunits of an RNR from subclass NrdAi, as well as the αβ octamer that forms in the presence of dATP. The results reveal a type of oligomer not previously seen for any class of RNR and suggest a mechanism for how binding of dATP to the ATP-cone switches off catalysis by sterically preventing formation of the asymmetrical αβ complex.
History
DepositionMay 21, 2021-
Header (metadata) releaseOct 6, 2021-
Map releaseOct 6, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12985.png

Downloads & links

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Map

FileDownload / File: emd_12985.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.088234216 - 0.1900865
Average (Standard dev.)-0.0007304346 (±0.009426957)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 367.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z448448448
origin x/y/z0.0000.0000.000
length x/y/z367.360367.360367.360
α/β/γ90.00090.00090.000
start NX/NY/NZ29290
NX/NY/NZ140137200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS448448448
D min/max/mean-0.0880.190-0.001

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Supplemental data

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Mask #1

Fileemd_12985_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : dATP-inhibited complex of NrdA and NrdB subunits from Leeuwenhoek...

EntireName: dATP-inhibited complex of NrdA and NrdB subunits from Leeuwenhoekiella blandensis
Components
  • Complex: dATP-inhibited complex of NrdA and NrdB subunits from Leeuwenhoekiella blandensis
    • Protein or peptide: Ribonucleotide reductase catalytic subunit from Leeuwenhoekiella blandensis
    • Protein or peptide: Ribonucleotide reductase radical generating subunit from Leeuwenhoekiella blandensis

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Supramolecule #1: dATP-inhibited complex of NrdA and NrdB subunits from Leeuwenhoek...

SupramoleculeName: dATP-inhibited complex of NrdA and NrdB subunits from Leeuwenhoekiella blandensis
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The complex was formed by incubating the NrdA and NrdB components in the presence of 2mM dATP followed by glutaraldehyde crosslinking on a gel filtration column.
Source (natural)Organism: Leeuwenhoekiella blandensis (bacteria)
Molecular weightTheoretical: 490 KDa

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Macromolecule #1: Ribonucleotide reductase catalytic subunit from Leeuwenhoekiella ...

MacromoleculeName: Ribonucleotide reductase catalytic subunit from Leeuwenhoekiella blandensis
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ribonucleoside-diphosphate reductase
Source (natural)Organism: Leeuwenhoekiella blandensis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MRENTTKLEN STETNVQHTK DNDLLKARRD ALQDATEKNT DEPSFDWLTE HSRSFLAAGY LSEGVSAEER IREIADRAE EILRMPGFSD KFYKYMGEGY FSLASPVWSN FGKKRGLPIS CFGSHIDDDM GNILYTQSEV G MMSKLGGG ...String:
MGSSHHHHHH SSGLVPRGSH MRENTTKLEN STETNVQHTK DNDLLKARRD ALQDATEKNT DEPSFDWLTE HSRSFLAAGY LSEGVSAEER IREIADRAE EILRMPGFSD KFYKYMGEGY FSLASPVWSN FGKKRGLPIS CFGSHIDDDM GNILYTQSEV G MMSKLGGG TSGYFGKIRH RGAAVKNNGY ASGAVHIMQL FDKMVDVVSQ GSVRRGRFSP YLPISHPDIK EF LEIGTEG NSIQQLTHGV TVDSTWMQEM IDGDTDKREV WAKVLQRRGE MGYPYIFYTD NANNGKPDVY KDK GHDIYA SNLCTEIMLP SSDEWSFVCV LSSINVLHYD KWKNTDAVET MVCFLDAVLT EFIDKLEEYR DSDN RDHRQ TFMFMERAYN FAKSNRALGL GVLGWHSLLQ SKRHAFDSQE AYDLNSEIFR EIKQRSYKAS EELAE KFGE PETLKGYGRR NATLNAIAPT TSSAFILGQV SQGIEPIWSN VYVKDIAKIK TTIKNPFLEE LFEEKG MNT PEVWRSVRDN DGSVQHLEFL TEQEKDVFKT YAEIDQMAII YQAANRQNHI DQGQSINLLV HPDMPIK EI NKIHITAWKL GLKSLYYQHS MNAAQKFKQK KECVSCEA

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Macromolecule #2: Ribonucleotide reductase radical generating subunit from Leeuwenh...

MacromoleculeName: Ribonucleotide reductase radical generating subunit from Leeuwenhoekiella blandensis
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Leeuwenhoekiella blandensis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSSQEIKKII KRDYSTAPFV LEKITNAIAN AMAALGHGSE QDAKLISMQV YESLLNNKEQ ESEYIPTVE QVQDMVEDKL MSSEFHDVAK AYIIYRNKRA LERKTNIFEK RINLKPYEYP E LNEYVAAI RHSYWIHTEF NFTSDIQDFK TGLSEVERSA IKNTMLAISQ ...String:
MSSQEIKKII KRDYSTAPFV LEKITNAIAN AMAALGHGSE QDAKLISMQV YESLLNNKEQ ESEYIPTVE QVQDMVEDKL MSSEFHDVAK AYIIYRNKRA LERKTNIFEK RINLKPYEYP E LNEYVAAI RHSYWIHTEF NFTSDIQDFK TGLSEVERSA IKNTMLAISQ IEVAVKTFWG DV HHRLPKP EIAAVGATFA ESEVRHHDAY SHLLEILGLN EEFKELKKKP VIMKRVHYLE TSL KHAKSD DDREYTESIL LFALFIEHVS LFSQFLIIMA FNKHKNMLKG ISNAVEATSK EEQI HGDFG VDIINIIKKE NPEWFDEEHN NLIKEMCLNS FEAESKVVDW IFEKGELDFL PKAVI NEFL KNRFNKSLEA IGLEKLFDID EALLQETEWF DDEIIGTKHG DFFVKRSINY SKRTQS ITS DDLF

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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

BufferpH: 7.6
Component:
ConcentrationFormulaName
50.0 mMHEPESHEPES buffer
50.0 mMNaClsodium chloride
2.0 % (v/v)glycerolglycerol
2.0 mMMgCl2magnesium chloride
2.0 mMTCEP
0.4 mMdATPdeoxyadenosine triphosphate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blot time 5 s, blot force -5, wait time 1 s, drain time 0 s.
DetailsThe specimen was taken directly from a fraction of a size exclusion chromatography run done in the presence of glutaraldehyde.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 493 / Average exposure time: 4.0 sec. / Average electron dose: 47.1 e/Å2
Details: Data recorded at the SciLifeLab facility in Umea, Sweden
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -0.003 µm / Nominal defocus min: -0.0015 µm / Nominal magnification: 165000
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: 200948
Details: Template-based picking using a docking model of the complex
CTF correctionSoftware - Name: cryoSPARC (ver. 2.15) / Software - details: Patch CTF
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 29902
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final 3D classificationNumber classes: 3 / Avg.num./class: 1 / Software - Name: cryoSPARC (ver. 2.15)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5olk

chain_id: A

5olk

chain_id: B

5olk

chain_id: C

5olk

chain_id: D
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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