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- EMDB-5945: Pyruvate Carboxylase tetramer in asymmetric architecture -

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Basic information

Entry
Database: EMDB / ID: EMD-5945
TitlePyruvate Carboxylase tetramer in asymmetric architecture
Map dataCryoEM of tetrameric Pyruvate Carboxylase in asymmetric architecture. Calculated after sorting of a catalytically active population.
Sample
  • Sample: Pyruvate Carboxylase
  • Protein or peptide: pyruvate carboxylate
Keywordsenzyme / tetrameric / biotin carboxylase
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.5 Å
AuthorsLasso G / Yu LPC / Gil D / Lazaro M / Tong L / Valle M
CitationJournal: Structure / Year: 2014
Title: Functional conformations for pyruvate carboxylase during catalysis explored by cryoelectron microscopy.
Authors: Gorka Lasso / Linda P C Yu / David Gil / Melisa Lázaro / Liang Tong / Mikel Valle /
Abstract: The tetrameric enzyme pyruvate carboxylase (PC), a biotin-dependent carboxylase, produces oxaloacetate by two consecutive reactions that take place in distant active sites. Previous crystal ...The tetrameric enzyme pyruvate carboxylase (PC), a biotin-dependent carboxylase, produces oxaloacetate by two consecutive reactions that take place in distant active sites. Previous crystal structures revealed two different configurations for PC tetramers, the so-called symmetric and asymmetric, which were understood as characteristic molecular architectures for PC from different organisms. We have analyzed PC samples from Staphylococcus aureus while the enzyme generates oxaloacetate, expecting PC tetramers to display the conformational landscape relevant for its functioning. Using cryoelectron microscopy (cryo-EM) and sorting techniques, we detect previously defined symmetric and asymmetric architectures, demonstrating that PC maps both arrangements by large conformational changes. Furthermore, we observe that each configuration is coupled to one of the two consecutive enzymatic reactions. The findings describe the structural transitions relevant for the allosteric control of the multifunctional PC and demonstrate that by cryo-EM and classification, we can characterize freely working macromolecules.
History
DepositionApr 10, 2014-
Header (metadata) releaseMay 14, 2014-
Map releaseAug 27, 2014-
UpdateAug 27, 2014-
Current statusAug 27, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_5945.map.gz / Format: CCP4 / Size: 25.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM of tetrameric Pyruvate Carboxylase in asymmetric architecture. Calculated after sorting of a catalytically active population.
Voxel sizeX=Y=Z: 1.75 Å
Density
Contour LevelBy AUTHOR: 0.005 / Movie #1: 0.005
Minimum - Maximum-0.000633 - 0.015584
Average (Standard dev.)0.00025733 (±0.00135165)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions190190190
Spacing190190190
CellA=B=C: 332.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.751.751.75
M x/y/z190190190
origin x/y/z0.0000.0000.000
length x/y/z332.500332.500332.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS190190190
D min/max/mean-0.0010.0160.000

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Supplemental data

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Sample components

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Entire : Pyruvate Carboxylase

EntireName: Pyruvate Carboxylase
Components
  • Sample: Pyruvate Carboxylase
  • Protein or peptide: pyruvate carboxylate

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Supramolecule #1000: Pyruvate Carboxylase

SupramoleculeName: Pyruvate Carboxylase / type: sample / ID: 1000 / Oligomeric state: Homotetramer / Number unique components: 1
Molecular weightTheoretical: 520 KDa

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Macromolecule #1: pyruvate carboxylate

MacromoleculeName: pyruvate carboxylate / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 520 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 Star (DE3) / Recombinant plasmid: pET28a

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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

Concentration0.05 mg/mL
BufferpH: 7.5
Details: 16.6 mM Tris-HCl, 1.7 mM acetyl-CoA, 15.7 mM pyruvate, 4.1 mM MgCl2, 1.7 mM DTT, 166.6 mM NaCl, 8.3 mM KHCO3, 1.66 mM ATP.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeJEOL 2200FSC
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 40000
Specialist opticsEnergy filter - Name: Omega in-column
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateJan 1, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 50 / Average electron dose: 15 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: Volume correction in defocus groups
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: OTHER / Software - Name: Relion, Spider / Number images used: 14032

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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