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- PDB-7zz3: Cryo-EM structure of "BC react" conformation of Lactococcus lacti... -

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Basic information

Entry
Database: PDB / ID: 7zz3
TitleCryo-EM structure of "BC react" conformation of Lactococcus lactis pyruvate carboxylase with acetyl-CoA
ComponentsPyruvate carboxylase
KeywordsLIGASE / Tetramer / carboxylase / biotin
Function / homology
Function and homology information


pyruvate carboxylase / pyruvate carboxylase activity / pyruvate metabolic process / gluconeogenesis / ATP binding / metal ion binding
Similarity search - Function
Pyruvate carboxylase / Carboxylase, conserved domain / Conserved carboxylase domain / Pyruvate carboxyltransferase / HMGL-like / Pyruvate carboxyltransferase domain. / Biotin carboxylase-like, N-terminal domain / Biotin carboxylase, C-terminal / Biotin carboxylation domain / Biotin carboxylase, N-terminal domain ...Pyruvate carboxylase / Carboxylase, conserved domain / Conserved carboxylase domain / Pyruvate carboxyltransferase / HMGL-like / Pyruvate carboxyltransferase domain. / Biotin carboxylase-like, N-terminal domain / Biotin carboxylase, C-terminal / Biotin carboxylation domain / Biotin carboxylase, N-terminal domain / Biotin carboxylase C-terminal domain / Biotin carboxylation domain profile. / Biotin carboxylase C-terminal domain / Carbamoyl-phosphate synthase subdomain signature 1. / Carbamoyl-phosphate synthetase large subunit-like, ATP-binding domain / Carbamoyl-phosphate synthase L chain, ATP binding domain / Biotin-requiring enzyme / Biotinyl/lipoyl domain profile. / Biotin/lipoyl attachment / Rudiment single hybrid motif / Single hybrid motif / Pre-ATP-grasp domain superfamily / ATP-grasp fold / ATP-grasp fold profile. / Carbamoyl-phosphate synthase subdomain signature 2. / Aldolase-type TIM barrel
Similarity search - Domain/homology
ACETYL COENZYME *A / ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / BICARBONATE ION / BIOTIN / : / PYRUVIC ACID / Pyruvate carboxylase
Similarity search - Component
Biological speciesLactococcus lactis (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.41 Å
AuthorsLopez-Alonso, J.P. / Lazaro, M. / Gil, D. / Choi, P.H. / Tong, L. / Valle, M.
Funding support France, Spain, 2items
OrganizationGrant numberCountry
Human Frontier Science Program (HFSP)RGP0062/2016 France
Spanish Ministry of Science, Innovation, and UniversitiesPGC2018-098996-B-100 Spain
CitationJournal: Nat Commun / Year: 2022
Title: CryoEM structural exploration of catalytically active enzyme pyruvate carboxylase.
Authors: Jorge Pedro López-Alonso / Melisa Lázaro / David Gil-Cartón / Philip H Choi / Alexandra Dodu / Liang Tong / Mikel Valle /
Abstract: Pyruvate carboxylase (PC) is a tetrameric enzyme that contains two active sites per subunit that catalyze two consecutive reactions. A mobile domain with an attached prosthetic biotin links both ...Pyruvate carboxylase (PC) is a tetrameric enzyme that contains two active sites per subunit that catalyze two consecutive reactions. A mobile domain with an attached prosthetic biotin links both reactions, an initial biotin carboxylation and the subsequent carboxyl transfer to pyruvate substrate to produce oxaloacetate. Reaction sites are at long distance, and there are several co-factors that play as allosteric regulators. Here, using cryoEM we explore the structure of active PC tetramers focusing on active sites and on the conformational space of the oligomers. The results capture the mobile domain at both active sites and expose catalytic steps of both reactions at high resolution, allowing the identification of substrates and products. The analysis of catalytically active PC tetramers reveals the role of certain motions during enzyme functioning, and the structural changes in the presence of additional cofactors expose the mechanism for allosteric regulation.
History
DepositionMay 25, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 12, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 26, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection / Refinement description
Category: atom_site / chem_comp_atom ...atom_site / chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pyruvate carboxylase
B: Pyruvate carboxylase
C: Pyruvate carboxylase
D: Pyruvate carboxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)515,49730
Polymers509,3984
Non-polymers6,09926
Water2,990166
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Pyruvate carboxylase /


Mass: 127349.578 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis (lactic acid bacteria)
Gene: BW154_09950 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2A9IR05, pyruvate carboxylase

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Non-polymers , 9 types, 192 molecules

#2: Chemical ChemComp-BTN / BIOTIN / Biotin


Mass: 244.311 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N2O3S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn
#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: Chemical
ChemComp-ACO / ACETYL COENZYME *A / Acetyl-CoA


Mass: 809.571 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C23H38N7O17P3S
#7: Chemical
ChemComp-PYR / PYRUVIC ACID / Pyruvic acid


Mass: 88.062 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H4O3
#8: Chemical ChemComp-BCT / BICARBONATE ION / Bicarbonate


Mass: 61.017 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: CHO3 / Comment: pH buffer*YM
#9: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#10: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 166 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Pyruvate carboxylase with acetyl coenzyme A / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Lactococcus lactis (lactic acid bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
220 mMTris hydrochlorideTris-HClTris1
35 mMmagnesium chlorideMgCl21
42 mMDithiothreitolDTT1
52 mMacetyl coenzyme AAcetyl-CoA1
620 mMpyruvatePyruvic acid1
72 mMadenosine triphosphateATPAdenosine triphosphate1
810 mMpotassium bicarbonateKHCO31
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 3.99 sec. / Electron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10518

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.0.6particle selection
2EPU2.4.0.54image acquisition
4CTFFIND4.1.5CTF correction
7PHENIX1.18.2model fitting
9PHENIX1.18.2model refinement
10RELION3.06initial Euler assignment
11RELION3.1.0final Euler assignment
13RELION3.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4578464
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128647 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5VYZ

5vyz


Accession code: 5VYZ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00334936
ELECTRON MICROSCOPYf_angle_d0.69647316
ELECTRON MICROSCOPYf_dihedral_angle_d6.6844807
ELECTRON MICROSCOPYf_chiral_restr0.0465213
ELECTRON MICROSCOPYf_plane_restr0.0056184

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