+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5945 | |||||||||
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Title | Pyruvate Carboxylase tetramer in asymmetric architecture | |||||||||
Map data | CryoEM of tetrameric Pyruvate Carboxylase in asymmetric architecture. Calculated after sorting of a catalytically active population. | |||||||||
Sample |
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Keywords | enzyme / tetrameric / biotin carboxylase | |||||||||
Biological species | Staphylococcus aureus (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 11.5 Å | |||||||||
Authors | Lasso G / Yu LPC / Gil D / Lazaro M / Tong L / Valle M | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5945.map.gz | 2.5 MB | EMDB map data format | |
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Header (meta data) | emd-5945-v30.xml emd-5945.xml | 9.9 KB 9.9 KB | Display Display | EMDB header |
Images | 400_5945.gif 80_5945.gif | 49.6 KB 3.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5945 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5945 | HTTPS FTP |
-Validation report
Summary document | emd_5945_validation.pdf.gz | 78.6 KB | Display | EMDB validaton report |
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Full document | emd_5945_full_validation.pdf.gz | 77.7 KB | Display | |
Data in XML | emd_5945_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5945 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5945 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5945.map.gz / Format: CCP4 / Size: 25.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | CryoEM of tetrameric Pyruvate Carboxylase in asymmetric architecture. Calculated after sorting of a catalytically active population. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.75 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Pyruvate Carboxylase
Entire | Name: Pyruvate Carboxylase |
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Components |
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-Supramolecule #1000: Pyruvate Carboxylase
Supramolecule | Name: Pyruvate Carboxylase / type: sample / ID: 1000 / Oligomeric state: Homotetramer / Number unique components: 1 |
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Molecular weight | Theoretical: 520 KDa |
-Macromolecule #1: pyruvate carboxylate
Macromolecule | Name: pyruvate carboxylate / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: Yes |
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Source (natural) | Organism: Staphylococcus aureus (bacteria) |
Molecular weight | Theoretical: 520 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 Star (DE3) / Recombinant plasmid: pET28a |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.05 mg/mL |
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Buffer | pH: 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. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK II |
-Electron microscopy
Microscope | JEOL 2200FSC |
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Specialist optics | Energy filter - Name: Omega in-column |
Date | Jan 1, 2011 |
Image recording | Category: 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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 40000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
CTF correction | Details: Volume correction in defocus groups |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: OTHER / Software - Name: Relion, Spider / Number images used: 14032 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: MDFF |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |