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- EMDB-5436: dATP-inhibited class Ia ribonucleotide reductase from E. coli: al... -

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Basic information

Entry
Database: EMDB / ID: EMD-5436
TitledATP-inhibited class Ia ribonucleotide reductase from E. coli: alpha4beta4 open conformation 6
Map dataalpha4beta4 open conformation 6
Sample
  • Sample: E. coli Class Ia ribonucleotide reductase
  • Protein or peptide: E. coli Class Ia ribonucleotide reductase
Keywordsribonucleotide reductase / allostery / inhibition / nucleotide metabolism
Function / homologyribonucleoside-diphosphate reductase complex
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 29.3 Å
AuthorsZimanyi CM / Ando N / Brignole E / Asturias FJ / Stubbe J / Drennan CL
CitationJournal: Structure / Year: 2012
Title: Tangled up in knots: structures of inactivated forms of E. coli class Ia ribonucleotide reductase.
Authors: Christina M Zimanyi / Nozomi Ando / Edward J Brignole / Francisco J Asturias / Joanne Stubbe / Catherine L Drennan /
Abstract: Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that ...Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.
History
DepositionJun 13, 2012-
Header (metadata) releaseJul 13, 2012-
Map releaseJul 25, 2012-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.83
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.83
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5436_1.png

Downloads & links

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Map

FileDownload / File: emd_5436.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationalpha4beta4 open conformation 6
Voxel sizeX: 4.36 Å / Y: 4.36 Å / Z: 6.54 Å
Density
Contour LevelBy AUTHOR: 1.83 / Movie #1: 1.83
Minimum - Maximum-5.86449909 - 11.91737556
Average (Standard dev.)0.00207146 (±0.74542516)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA: 313.92 Å / B: 313.92 Å / C: 470.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.366.54
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z313.920313.920470.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-5.86411.9170.002

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Supplemental data

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Sample components

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Entire : E. coli Class Ia ribonucleotide reductase

EntireName: E. coli Class Ia ribonucleotide reductase
Components
  • Sample: E. coli Class Ia ribonucleotide reductase
  • Protein or peptide: E. coli Class Ia ribonucleotide reductase

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Supramolecule #1000: E. coli Class Ia ribonucleotide reductase

SupramoleculeName: E. coli Class Ia ribonucleotide reductase / type: sample / ID: 1000
Oligomeric state: two alpha2 subunits in complex with two beta2 subunits
Number unique components: 1
Molecular weightExperimental: 517 KDa / Theoretical: 517 KDa / Method: Calculated from amino acid sequence of subunits

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Macromolecule #1: E. coli Class Ia ribonucleotide reductase

MacromoleculeName: E. coli Class Ia ribonucleotide reductase / type: protein_or_peptide / ID: 1 / Name.synonym: RNR / Details: alpha4beta4 open conformation / Number of copies: 1 / Oligomeric state: hetero-octamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 517 KDa / Theoretical: 517 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: ribonucleoside-diphosphate reductase complex

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
Details: 50 mM HEPES, 15 mM MgCl2, 1 mM EDTA, 1 mM CDP, 0.05 mM dATP
StainingType: NEGATIVE
Details: 5ul protein, washed immediately 3x [2% uranyl acetate, 0.2% trehalose], carbon sandwich
GridDetails: thin carbon support on 300 mesh Cu/Rh grid, glow discharge in amylamine
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.801 µm / Nominal defocus min: 0.275 µm / Nominal magnification: 50000
Sample stageSpecimen holder: room temperature / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -59.1 / Tilt angle max: -54.3
DateJul 31, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 89 / Average electron dose: 25 e/Å2 / Details: images acquired as tilt-pairs / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 29.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 385
DetailsSemi-automated particle selection from untilted images with EMAN2. Particles matched in tilted images using modified TiltPicker. Processed in SPIDER.

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Atomic model buiding 1

Initial modelPDB ID:

4r1r


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: D / Chain - #3 - Chain ID: E
SoftwareName: Chimera
DetailsProtocol: rigid body. Subunits iteratively fit with Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation

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Atomic model buiding 2

Initial modelPDB ID:

1rib


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsProtocol: rigid body. Subunits iteratively fit with Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation

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