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- EMDB-1744: Pyruvate carboxylase from S. aureus after addition of acetyl-CoA,... -

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Basic information

Entry
Database: EMDB / ID: EMD-1744
TitlePyruvate carboxylase from S. aureus after addition of acetyl-CoA, AMP-PNP, KHCO3 and oxaloacetate (based on a subset obtained after maximum likelihood based classification)
Map dataPyruvate carboxylase from S. aureus after addition of acetyl-CoA, AMP-PNP, KHCO3 and oxaloacetate (based on a subset obtained after maximum likelihood based classification)
Sample
  • Sample: Pyruvate carboxylase from S. aureus after addition of acetyl-CoA, AMP-PNP, KHCO3 and oxaloacetate (based on a subset obtained after maximum likelihood based classification)
  • Protein or peptide: Pyruvate carboxylase
KeywordsPyruvate carboxylase / Biotin-dependent / Acetyl-CoA / Multifunctional / Pyruvate / Oxaloacetate / AMP-PNP / EC 6.4.1.1 / Ligase / Maximum likelihood based classification
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.5 Å
AuthorsLasso G / Yu LPC / Gil D / Xiang S / Tong L / Valle M
CitationJournal: Structure / Year: 2010
Title: Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase.
Authors: Gorka Lasso / Linda P C Yu / David Gil / Song Xiang / Liang Tong / Mikel Valle /
Abstract: Pyruvate carboxylase (PC) is a conserved multifunctional enzyme linked to important metabolic diseases. PC homotetramer is arranged in two layers with two opposing monomers per layer. Cryo-EM ...Pyruvate carboxylase (PC) is a conserved multifunctional enzyme linked to important metabolic diseases. PC homotetramer is arranged in two layers with two opposing monomers per layer. Cryo-EM explores the conformational variability of PC in the presence of different substrates. The results demonstrate that the biotin-carboxyl carrier protein (BCCP) domain localizes near the biotin carboxylase (BC) domain of its own monomer and travels to the carboxyltransferase (CT) domain of the opposite monomer. All density maps show noticeable conformational differences between layers, mainly for the BCCP and BC domains. This asymmetry may be indicative of a coordination mechanism where monomers from different layers catalyze the BC and CT reactions consecutively. A conformational change of the PC tetramerization (PT) domain suggests a new functional role in communication. A long-range communication pathway between subunits in different layers, via interacting PT-PT and BC-BC domains, may be responsible for the cooperativity of PC from Staphylococcus aureus.
History
DepositionJun 2, 2010-
Header (metadata) releaseJun 11, 2010-
Map releaseMay 20, 2011-
UpdateMay 20, 2011-
Current statusMay 20, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.76
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.76
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1744.map.gz / Format: CCP4 / Size: 25.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPyruvate carboxylase from S. aureus after addition of acetyl-CoA, AMP-PNP, KHCO3 and oxaloacetate (based on a subset obtained after maximum likelihood based classification)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.75 Å/pix.
x 190 pix.
= 332.5 Å
1.75 Å/pix.
x 190 pix.
= 332.5 Å
1.75 Å/pix.
x 190 pix.
= 332.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.75 Å
Density
Contour LevelBy AUTHOR: 3.76 / Movie #1: 3.76
Minimum - Maximum-3.95412 - 11.028
Average (Standard dev.)0.103154 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions190190190
Spacing190190190
CellA=B=C: 332.5 Å
α=β=γ: 90 °

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/NZ-100-100-100
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS190190190
D min/max/mean-3.95411.0280.103

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Supplemental data

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Sample components

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Entire : Pyruvate carboxylase from S. aureus after addition of acetyl-CoA,...

EntireName: Pyruvate carboxylase from S. aureus after addition of acetyl-CoA, AMP-PNP, KHCO3 and oxaloacetate (based on a subset obtained after maximum likelihood based classification)
Components
  • Sample: Pyruvate carboxylase from S. aureus after addition of acetyl-CoA, AMP-PNP, KHCO3 and oxaloacetate (based on a subset obtained after maximum likelihood based classification)
  • Protein or peptide: Pyruvate carboxylase

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Supramolecule #1000: Pyruvate carboxylase from S. aureus after addition of acetyl-CoA,...

SupramoleculeName: Pyruvate carboxylase from S. aureus after addition of acetyl-CoA, AMP-PNP, KHCO3 and oxaloacetate (based on a subset obtained after maximum likelihood based classification)
type: sample / ID: 1000 / Oligomeric state: Homotetramer / Number unique components: 1

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Macromolecule #1: Pyruvate carboxylase

MacromoleculeName: Pyruvate carboxylase / type: protein_or_peptide / ID: 1 / Name.synonym: PC / Oligomeric state: Homotetramer / Recombinant expression: Yes
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 520 KDa
Recombinant expressionOrganism: Escherichia coli BL21 Star / Recombinant plasmid: pET28a

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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: 7.5
Details: 20mM Tris HCl, 2mM NaCl, 2mM DTT, 2mM acetyl-CoA, 2mM AMP-PNP, 50mM KHCO3, 10mM oxaloacetate
StainingType: NEGATIVE / Details: Cryo-EM
GridDetails: Quantifoil R2/2, 100 holey carbon films, cu 200 mesh
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot (FEI) / Timed resolved state: 45 sec / Method: Blot for 1.5 seconds

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Electron microscopy

MicroscopeJEOL 2200FS
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
Sample stageSpecimen holder: Single tilt cryoholder / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 99 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 1.75 µm / Average electron dose: 11 e/Å2

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Image processing

CTF correctionDetails: By defocus groups (Wiener filter)
Final angle assignmentDetails: spider
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: OTHER / Software - Name: Spider xmipp
Details: The map was obtained based on a subset of particles obtained after maximum likelihood classification
Number images used: 27943

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