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- EMDB-1807: Saccharomyces cerevisiae ribonucleotide reductase hole complex at... -

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Basic information

Entry
Database: EMDB / ID: EMD-1807
TitleSaccharomyces cerevisiae ribonucleotide reductase hole complex at the presence of dATP
Map dataThis is an EM map of Yeast dATP Ribonucleotide Reductase complex
Sample
  • Sample: Yeast ribonucleotide reductase complex
  • Protein or peptide: Yeast ribonucleotide reductase RR1
  • Protein or peptide: Yeast ribonucleotide reductase RR2.RR4
KeywordsRibonucleotide reductase / hexamer / dimer
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 28.0 Å
AuthorsFairman JW / Wijerathna SR / Ahmad MF / Xu H / Nakano R / Jha S / Prendergast J / Welin RM / Flodin S / Roos A ...Fairman JW / Wijerathna SR / Ahmad MF / Xu H / Nakano R / Jha S / Prendergast J / Welin RM / Flodin S / Roos A / Nordlund P / Li Z / Walz T / Dealwis CG
CitationJournal: Nat Struct Mol Biol / Year: 2011
Title: Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization.
Authors: James Wesley Fairman / Sanath Ranjan Wijerathna / Md Faiz Ahmad / Hai Xu / Ryo Nakano / Shalini Jha / Jay Prendergast / R Martin Welin / Susanne Flodin / Annette Roos / Pär Nordlund / ...Authors: James Wesley Fairman / Sanath Ranjan Wijerathna / Md Faiz Ahmad / Hai Xu / Ryo Nakano / Shalini Jha / Jay Prendergast / R Martin Welin / Susanne Flodin / Annette Roos / Pär Nordlund / Zongli Li / Thomas Walz / Chris Godfrey Dealwis /
Abstract: Ribonucleotide reductase (RR) is an α(n)β(n) (RR1-RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required ...Ribonucleotide reductase (RR) is an α(n)β(n) (RR1-RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP-GDP, TTP-ATP, and TTP-dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1-dATP hexamer and used single-particle electron microscopy to visualize the α(6)-ββ'-dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.
History
DepositionOct 21, 2010-
Header (metadata) releaseDec 24, 2010-
Map releaseJul 19, 2011-
UpdateJul 19, 2011-
Current statusJul 19, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.268
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.268
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1807.tif

Downloads & links

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Map

FileDownload / File: emd_1807.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an EM map of Yeast dATP Ribonucleotide Reductase complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.1 Å/pix.
x 128 pix.
= 396.8 Å		3.1 Å/pix.
x 128 pix.
= 396.8 Å		3.1 Å/pix.
x 128 pix.
= 396.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.268 / Movie #1: 0.268
Minimum - Maximum-0.131972 - 0.55113
Average (Standard dev.)0.012938 (±0.0596164)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-4-4-4
Dimensions128128128
Spacing128128128
CellA=B=C: 396.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.13.13.1
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z396.800396.800396.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-150-149
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS-4-4-4
NC/NR/NS128128128
D min/max/mean-0.1320.5510.013

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Supplemental data

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Sample components

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Entire : Yeast ribonucleotide reductase complex

EntireName: Yeast ribonucleotide reductase complex
Components
  • Sample: Yeast ribonucleotide reductase complex
  • Protein or peptide: Yeast ribonucleotide reductase RR1
  • Protein or peptide: Yeast ribonucleotide reductase RR2.RR4

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Supramolecule #1000: Yeast ribonucleotide reductase complex

SupramoleculeName: Yeast ribonucleotide reductase complex / type: sample / ID: 1000
Oligomeric state: One homohexamer of yeast RR1 binds to one hetero-dimer of yeast RR2.RR4
Number unique components: 2
Molecular weightExperimental: 700 KDa / Theoretical: 680 KDa / Method: Size exclusion chromatography

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Macromolecule #1: Yeast ribonucleotide reductase RR1

MacromoleculeName: Yeast ribonucleotide reductase RR1 / type: protein_or_peptide / ID: 1 / Name.synonym: RR1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast

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Macromolecule #2: Yeast ribonucleotide reductase RR2.RR4

MacromoleculeName: Yeast ribonucleotide reductase RR2.RR4 / type: protein_or_peptide / ID: 2 / Name.synonym: RR2.RR4 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8.5
Details: 50mM Ammonium actate,5mM MgCl2, 0.1M KCl, 50uM dATP,100uM hydroxyurea with 5% glycerol
StainingType: NEGATIVE
Details: Grids with adsorbed protein was stained on 0.75% w/v uranyl formate for 30 seconds.
GridDetails: Quantifoil R2/1 Coppor grid
VitrificationCryogen name: NITROGEN / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnificatioin
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 4.2 µm / Number real images: 52
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49883 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 50
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe quantifoil grids are pre-coated with continuous carbon film. 5 ul sample is applied onto the carbon film grid,wait for 30 seconds, blot from side, wash it in a drop of water, blot from side, stain in a drop of 0.75% uranyl formate for 30 seconds, with the sample side facing up insert into a drop of 0.75% uranyl formate where a small piece of carbon film has been floated, pick up the carbon film from underneath, gently blot from both side. Monitor the thickness of the remaining sample between two carbon films . When the thickness is becoming right (milky in color), plunge it into liquid nitrogen to freeze. Look at the grid with cryo EM procedure.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: The final map was calculated from a selected sub-data-set based on 2D classification using Back Projection and Angular Refinement routines in Spider.
Number images used: 829
Final two d classificationNumber classes: 50

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Atomic model buiding 1

Initial modelPDB ID:

1jk0

SoftwareName: Camera
DetailsProtocol: Rigid Body. The Yeast RR1.dATP hexamer (3PAW) was fitted into the EM density map and the Yeast RR2.RR4 heterodimer (1JK0) was fitted into the difference map using camera fit in map function.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

3paw

SoftwareName: Camera
DetailsProtocol: Rigid Body. The Yeast RR1.dATP hexamer (3PAW) was fitted into the EM density map and the Yeast RR2.RR4 heterodimer (1JK0) was fitted into the difference map using camera fit in map function.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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