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- EMDB-1527: The structure of phosphorylase kinase holoenzyme at 9.9 A resolut... -

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Basic information

Entry
Database: EMDB / ID: EMD-1527
TitleThe structure of phosphorylase kinase holoenzyme at 9.9 A resolution and location of the catalytic subunit and the substrate glycogen phosphorylase
Map data3D map of PhK
Sample
  • Sample: Phosphorylase kinase holoenzyme purified from rabbit muscle
  • Protein or peptide: Phosphorylase kinase
KeywordsPhosphorylase kinase
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.9 Å
AuthorsVenien-Bryan C / Jonic S / Skamnaki V / Brown N / Bishler N / Oikonomakos NG / Boisset N / Johnson LN
CitationJournal: Structure / Year: 2009
Title: The structure of phosphorylase kinase holoenzyme at 9.9 angstroms resolution and location of the catalytic subunit and the substrate glycogen phosphorylase.
Authors: Catherine Vénien-Bryan / Slavica Jonic / Vasiliki Skamnaki / Nick Brown / Nicolas Bischler / Nikos G Oikonomakos / Nicolas Boisset / Louise N Johnson /
Abstract: Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), ...Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 angstroms resolution structure of PhK heterotetramer (alphabetagammadelta)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic gamma subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 angstroms resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 angstroms resolution was consistent with a proteolysed activated form of PhK that had lost the alpha subunits and possibly the gamma subunits.
History
DepositionJun 23, 2008-
Header (metadata) releaseJun 24, 2008-
Map releaseApr 2, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.001
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.001
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1527.gif

Downloads & links

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Map

FileDownload / File: emd_1527.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of PhK
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.07 Å/pix.
x 180 pix.
= 372.6 Å		2.07 Å/pix.
x 180 pix.
= 372.6 Å		2.07 Å/pix.
x 180 pix.
= 372.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.07 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.000757 / Movie #1: 0.001
Minimum - Maximum-0.0273353 - 0.0300857
Average (Standard dev.)0.0000545086 (±0.00140281)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 372.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.072.072.07
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z372.600372.600372.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-75-75-74
NX/NY/NZ150150150
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0270.0300.000

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Supplemental data

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Sample components

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Entire : Phosphorylase kinase holoenzyme purified from rabbit muscle

EntireName: Phosphorylase kinase holoenzyme purified from rabbit muscle
Components
  • Sample: Phosphorylase kinase holoenzyme purified from rabbit muscle
  • Protein or peptide: Phosphorylase kinase

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Supramolecule #1000: Phosphorylase kinase holoenzyme purified from rabbit muscle

SupramoleculeName: Phosphorylase kinase holoenzyme purified from rabbit muscle
type: sample / ID: 1000
Oligomeric state: hexadecamer assembly of four different subunits arranged as an (abgd)4 tetramer
Number unique components: 4
Molecular weightExperimental: 1.3 MDa / Theoretical: 1.3 MDa

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Macromolecule #1: Phosphorylase kinase

MacromoleculeName: Phosphorylase kinase / type: protein_or_peptide / ID: 1 / Name.synonym: PhK / Number of copies: 4 / Oligomeric state: hexadecamer / Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: muscle
Molecular weightExperimental: 1.3 MDa / Theoretical: 1.3 MDa

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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

Concentration1.0 mg/mL
BufferpH: 8.2
Details: 100mM NaCl, 0.3 mM CaCl2, 5mM MgCl2, 50 mM Hepes, pH 8.2
GridDetails: 400-mesh copper grid coated with a thin holey-carbon film
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Manual. 5 microL were applied on a 200 mesh copper grid, coated with a thin holey carbon film. After blotting the excess of solution with Whatman paper, the grid ...Details: Vitrification instrument: Manual. 5 microL were applied on a 200 mesh copper grid, coated with a thin holey carbon film. After blotting the excess of solution with Whatman paper, the grid was rapidly plunged into liquid ethane
Method: Single-sided blotting

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Electron microscopy

MicroscopeJEOL 2010UHR
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.5 mm / Nominal defocus max: 4.8 µm / Nominal defocus min: 2.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 91.15 K / Max: 93.15 K / Average: 93.15 K
Detailslow dose illumination
DateApr 13, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 2.07 µm / Number real images: 98 / Average electron dose: 10 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Wiener filtration of volumes from focal series
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Final map was calculated using five groups of defocus
Number images used: 18123
DetailsThe particles were selected using an automatic selection program

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Atomic model buiding 1

Initial modelPDB ID:

2phk

SoftwareName: Situs
DetailsProtocol: rigid body
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation coefficient

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