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- EMDB-15034: Cryo-EM structure of "BC closed" conformation of Lactococcus lact... -

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Basic information

Entry
Database: EMDB / ID: EMD-15034
TitleCryo-EM structure of "BC closed" conformation of Lactococcus lactis pyruvate carboxylase with acetyl-CoA
Map data
Sample
  • Organelle or cellular component: Pyruvate carboxylase with acetyl coenzyme A
    • Protein or peptide: Pyruvate carboxylase
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ACETYL COENZYME *A
  • Ligand: water
KeywordsTetramer / carboxylase / biotin / inhibitor / LIGASE
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 / Rudiment single hybrid motif / Biotinyl/lipoyl domain profile. / Biotin/lipoyl attachment / 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
Pyruvate carboxylase
Similarity search - Component
Biological speciesLactococcus lactis (lactic acid bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsLopez-Alonso JP / Lazaro M / Gil D / Choi PH / Tong L / Valle M
Funding support Spain, 2 items
OrganizationGrant numberCountry
Human Frontier Science Program (HFSP)RGP0062/2016 Spain
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, 2022-
Header (metadata) releaseOct 12, 2022-
Map releaseOct 12, 2022-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15034.png

Downloads & links

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Map

FileDownload / File: emd_15034.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 300 pix.
= 318. Å		1.06 Å/pix.
x 300 pix.
= 318. Å		1.06 Å/pix.
x 300 pix.
= 318. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-0.19392945 - 0.5615073
Average (Standard dev.)0.00032956532 (±0.00787052)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15034_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15034_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15034_half_map_2.map
Projections & Slices
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Sample components

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Entire : Pyruvate carboxylase with acetyl coenzyme A

EntireName: Pyruvate carboxylase with acetyl coenzyme A
Components
  • Organelle or cellular component: Pyruvate carboxylase with acetyl coenzyme A
    • Protein or peptide: Pyruvate carboxylase
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ACETYL COENZYME *A
  • Ligand: water

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Supramolecule #1: Pyruvate carboxylase with acetyl coenzyme A

SupramoleculeName: Pyruvate carboxylase with acetyl coenzyme A / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Lactococcus lactis (lactic acid bacteria)

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Macromolecule #1: Pyruvate carboxylase

MacromoleculeName: Pyruvate carboxylase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: pyruvate carboxylase
Source (natural)Organism: Lactococcus lactis (lactic acid bacteria)
Molecular weightTheoretical: 127.349578 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: VPRGSHMKKL LVANRGEIAV RVFRACNELG LSTVAVYARE DEYSVHRFKA DESYLIGQGK KPIDAYLDID DIIRVALESG ADAIHPGYG LLSENLEFAT KVRAAGLVFV GPELHHLDIF GDKIKAKAAA DEAKVPGIPG TNGAVDIDGA LEFAKTYGYP V MIKAALGG ...String:
VPRGSHMKKL LVANRGEIAV RVFRACNELG LSTVAVYARE DEYSVHRFKA DESYLIGQGK KPIDAYLDID DIIRVALESG ADAIHPGYG LLSENLEFAT KVRAAGLVFV GPELHHLDIF GDKIKAKAAA DEAKVPGIPG TNGAVDIDGA LEFAKTYGYP V MIKAALGG GGRGMRVARN DAEMHDGYAR AKSEAIGAFG SGEIYVEKYI ENPKHIEVQI LGDRHGNIIH LHERDCSVQR RN QKVIEIA PAVGLSPDFR NEICEAAVKL CKNVGYVNAG TVEFLVKDDK FYFIEVNPRV QVEHTITELI TGVDIVQAQI LIA QGKDLH REIGLPAQSE IPLLGSAIQC RITTEDPQNG FLPDTGKIDT YRSPGGFGIR LDVGNAYAGY EVTPYFDSLL VKVC TFANE FSDSVRKMDR VLHEFRIRGV KTNIPFLINV IANENFTSGQ ATTTFIDNTP SLFNFPRLRD RGTKTLHYLS MITVN GFPG IENTEKRHFE EPRQPLLNLE KKKTAKNILD EQGADAVVDY VKNTKEVLLT DTTLRDAHQS LLATRLRLQD MKGIAQ AID QGLPELFSAE MWGGATFDVA YRFLNESPWY RLRKLRKLMP NTMFQMLFRG SNAVGYQNYP DNVIEEFIRV AAHEGID VF RIFDSLNWLP QMEKSIQAVR DNGKIAEATI CYTGDILDPS RPKYNIQYYK DLAKELEATG AHILAV(KCX)DMA GLLK PQAAY RLISELKDTV DLPIHLHTHD TSGNGIITYS GATQAGVDII DVATASLAGG TSQPSMQSIY YALEHGPRHA SINVK NAEQ IDHYWEDVRK YYAPFEAGIT SPQTEVYMHE MPGGQYTNLK SQAAAVGLGH RFDEIKQMYR KVNMMFGDII KVTPSS KVV GDMALFMIQN DLTEEDVYAR GNELNFPESV VSFFRGDLGQ PVGGFPEKLQ KIIVKDKAVI TDRPGLHAEK VDFETVK AD LEQKIGYEPG DHEVISYIMY PQVFLDYQKM QREFGAVTLL DTPTFLHGMR LNEKIEVQIE KGKTLSIRLD EIGEPDLA G NRVLFFNLNG QRREVVINDQ SVQAQVVAKR KAETGNPNQI GATMPGSVLE ILVKAGDKVQ KGQALMVTEA MKMETTIEA PFDGEIVDLH VVKGEAIQTQ DLLIEIN

UniProtKB: Pyruvate carboxylase

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: ACETYL COENZYME *A

MacromoleculeName: ACETYL COENZYME *A / type: ligand / ID: 4 / Number of copies: 1 / Formula: ACO
Molecular weightTheoretical: 809.571 Da
Chemical component information

ChemComp-ACO:
ACETYL COENZYME *A

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 11 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state3D array

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
20.0 mMTris-HClTris hydrochloride
5.0 mMMgCl2magnesium chloride
2.0 mMDTTDithiothreitol
2.0 mMacetyl coenzyme A
20.0 mMpyruvate
2.0 mMATPadenosine triphosphate
10.0 mMKHCO3potassium bicarbonate
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 10518 / Average exposure time: 3.99 sec. / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 81000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4578464
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5vyw

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.06) / Number images used: 182007
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.06)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final 3D classificationNumber classes: 10 / Avg.num./class: 136909 / Software - Name: RELION (ver. 3.1.0) / Details: 3D classification of a BC domain
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5vyw


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7zz4:
Cryo-EM structure of "BC closed" conformation of Lactococcus lactis pyruvate carboxylase with acetyl-CoA

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