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- EMDB-5345: CryoEM Structure of the Human Propionyl-CoA Carboxylase -

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Basic information

Entry
Database: EMDB / ID: EMD-5345
TitleCryoEM Structure of the Human Propionyl-CoA Carboxylase
Map dataThis is the volume.
Sample
  • Sample: Human Propionyl-CoA Carboxylase holoenzyme
  • Protein or peptide: Human Propionyl-CoA Carboxylase
KeywordsCryoEM / human propionyl-CoA carboxylase / three-dimensional structure / single particle analysis
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsZhou ZH / Deng B / Shen Y / Tong L
CitationJournal: Nature / Year: 2010
Title: Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.
Authors: Christine S Huang / Kianoush Sadre-Bazzaz / Yang Shen / Binbin Deng / Z Hong Zhou / Liang Tong /
Abstract: Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd ...Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).
History
DepositionOct 4, 2011-
Header (metadata) releaseDec 1, 2011-
Map releaseDec 1, 2011-
UpdateDec 1, 2011-
Current statusDec 1, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.423
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.423
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5345_1.png

Downloads & links

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Map

FileDownload / File: emd_5345.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the volume.
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 0.423 / Movie #1: 0.423
Minimum - Maximum-3.60469 - 3.74047
Average (Standard dev.)-0.00000000126603 (±0.385184)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-56-56-56
Dimensions112112112
Spacing112112112
CellA=B=C: 313.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z313.600313.600313.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-56-56-56
NC/NR/NS112112112
D min/max/mean-3.6053.740-0.000

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Supplemental data

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Sample components

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Entire : Human Propionyl-CoA Carboxylase holoenzyme

EntireName: Human Propionyl-CoA Carboxylase holoenzyme
Components
  • Sample: Human Propionyl-CoA Carboxylase holoenzyme
  • Protein or peptide: Human Propionyl-CoA Carboxylase

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Supramolecule #1000: Human Propionyl-CoA Carboxylase holoenzyme

SupramoleculeName: Human Propionyl-CoA Carboxylase holoenzyme / type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: dodecamer (six alpha and six beta subunits)
Number unique components: 2
Molecular weightExperimental: 750 KDa / Theoretical: 768 KDa / Method: SDS PAGE

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Macromolecule #1: Human Propionyl-CoA Carboxylase

MacromoleculeName: Human Propionyl-CoA Carboxylase / type: protein_or_peptide / ID: 1 / Name.synonym: Human Propionyl-CoA Carboxylase / Details: PCC in solution / Number of copies: 6 / Oligomeric state: dodecamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytoplasm
Molecular weightExperimental: 750 KDa / Theoretical: 768 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET-26b

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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

Concentration0.07 mg/mL
BufferpH: 7.4 / Details: 25 mM Tris, 250 mM NaCl, 2 mM DTT, 5% v/v glycerol
GridDetails: 300 mesh copper quantifoil grid
VitrificationCryogen name: NITROGEN / Chamber humidity: 40 % / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home-made plunger / Method: blot for 2 seconds before plunging

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Electron microscopy

MicroscopeJEOL 1200EX
Electron beamAcceleration voltage: 100 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.1 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 77 K / Max: 100 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: corrected at 100,000 times mag
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Average electron dose: 10 e/Å2 / Bits/pixel: 12

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 1000

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