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- EMDB-1899: Reconstruction of the 3D model of AMPK trimer in AMP binding state. -

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Basic information

Entry
Database: EMDB / ID: EMD-1899
TitleReconstruction of the 3D model of AMPK trimer in AMP binding state.
Map dataThis is an image of a surface rendered side-view of AMP-activated protein kinase bound with AMP.
Sample
  • Sample: Full-Length Rat AMP-Activated Protein Kinase
  • Protein or peptide: Mammalian AMP-Activated Protein Kinase
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsZhu L / Chen L / Zhou XM / Zhang YY / Zhang YJ / Zhao J / Ji SR / Wu JW / Wu Y
CitationJournal: Structure / Year: 2011
Title: Structural insights into the architecture and allostery of full-length AMP-activated protein kinase.
Authors: Li Zhu / Lei Chen / Xiao-Ming Zhou / Yuan-Yuan Zhang / Yi-Jiong Zhang / Jing Zhao / Shang-Rong Ji / Jia-Wei Wu / Yi Wu /
Abstract: AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of α catalytic subunit, β scaffolding subunit, and γ regulatory subunit with critical roles in maintaining cellular energy ...AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of α catalytic subunit, β scaffolding subunit, and γ regulatory subunit with critical roles in maintaining cellular energy homeostasis. However, the molecular architecture of the intact complex and the allostery associated with the adenosine binding-induced regulation of kinase activity remain unclear. Here, we determine the three-dimensional reconstruction and subunit organization of the full-length rat AMPK (α1β1γ1) through single-particle electron-microscopy. By comparing the structures of AMPK in ATP- and AMP-bound states, we are able to visualize the sequential conformational changes underlying kinase activation that transmits from the adenosine binding sites in the γ subunit to the kinase domain of the α subunit. These results not only make substantial revision to the current model of AMPK assembly, but also highlight a central role of the linker sequence of the α subunit in mediating the allostery of AMPK.
History
DepositionMay 24, 2011-
Header (metadata) releaseMay 27, 2011-
Map releaseMay 27, 2011-
UpdateMay 27, 2011-
Current statusMay 27, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_1899.png

Downloads & links

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Map

FileDownload / File: emd_1899.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an image of a surface rendered side-view of AMP-activated protein kinase bound with AMP.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.23 Å/pix.
x 72 pix.
= 304.56 Å		4.23 Å/pix.
x 72 pix.
= 304.56 Å		4.23 Å/pix.
x 72 pix.
= 304.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.23 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-3.06265 - 12.136900000000001
Average (Standard dev.)-0.289054 (±0.999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 304.56 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.234.234.23
M x/y/z727272
origin x/y/z-0.000-0.000-0.000
length x/y/z304.560304.560304.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-3.06312.137-0.289

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Supplemental data

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Sample components

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Entire : Full-Length Rat AMP-Activated Protein Kinase

EntireName: Full-Length Rat AMP-Activated Protein Kinase
Components
  • Sample: Full-Length Rat AMP-Activated Protein Kinase
  • Protein or peptide: Mammalian AMP-Activated Protein Kinase

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Supramolecule #1000: Full-Length Rat AMP-Activated Protein Kinase

SupramoleculeName: Full-Length Rat AMP-Activated Protein Kinase / type: sample / ID: 1000 / Oligomeric state: homotrimer / Number unique components: 3
Molecular weightExperimental: 390 KDa / Theoretical: 390 KDa

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Macromolecule #1: Mammalian AMP-Activated Protein Kinase

MacromoleculeName: Mammalian AMP-Activated Protein Kinase / type: protein_or_peptide / ID: 1 / Name.synonym: AMPK / Number of copies: 3 / Oligomeric state: Homotrimer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norway Rat
Molecular weightExperimental: 390 KDa / Theoretical: 390 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.15 mg/mL
BufferpH: 6.8 / Details: 10mM Tris,15mM NaCl, 1mM TCEP,1.2mM MgCl2
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 30 seconds.
GridDetails: 300 mesh copper EM grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F30
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 21.17 µm / Number real images: 160 / Average electron dose: 18 e/Å2 / Bits/pixel: 16
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Single tilt / Specimen holder model: OTHER / Tilt angle min: -45
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsThe particle pairs were extracted using WEB program with a box size of 72 pixels.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: High-quality classes without significant differences in initial reconstructions were merged for calculating new volumes.
Number images used: 7602
Final angle assignmentDetails: SPIDER:theta 45 degrees
Final two d classificationNumber classes: 10

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Atomic model buiding 1

Initial model
PDB IDChain

3h4j

chain_id: B

2v9j


1z0m

chain_id: A
SoftwareName: SITUS,CHIMERA
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. The domains were separately fitted both by calculated and manual docking using program SITUS and CHIMERA.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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