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- PDB-7plm: CryoEM reconstruction of pyruvate ferredoxin oxidoreductase (PFOR... -

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Basic information

Entry
Database: PDB / ID: 7plm
TitleCryoEM reconstruction of pyruvate ferredoxin oxidoreductase (PFOR) in anaerobic conditions
ComponentsPyruvate:ferredoxin oxidoreductase
KeywordsOXIDOREDUCTASE / 4Fe-4S / anaerobic / PYRUVATE CATABOLISM / Electron Transport
Function / homology
Function and homology information


pyruvate synthase / pyruvate synthase activity / thiamine pyrophosphate binding / electron transport chain / 4 iron, 4 sulfur cluster binding / response to oxidative stress / iron ion binding / cytoplasm
Similarity search - Function
: / Pyruvate-ferredoxin oxidoreductase, insertion domain / Pyruvate-flavodoxin oxidoreductase / Pyruvate-flavodoxin oxidoreductase, EKR domain / Pyruvate-flavodoxin oxidoreductase, EKR domain superfamily / Domain of unknown function / Domain of unknown function / Pyruvate-ferredoxin Oxidoreductase; domain 3 / Pyruvate-ferredoxin oxidoreductase, PFOR, domain III / Pyruvate:ferredoxin oxidoreductase, core domain II ...: / Pyruvate-ferredoxin oxidoreductase, insertion domain / Pyruvate-flavodoxin oxidoreductase / Pyruvate-flavodoxin oxidoreductase, EKR domain / Pyruvate-flavodoxin oxidoreductase, EKR domain superfamily / Domain of unknown function / Domain of unknown function / Pyruvate-ferredoxin Oxidoreductase; domain 3 / Pyruvate-ferredoxin oxidoreductase, PFOR, domain III / Pyruvate:ferredoxin oxidoreductase, core domain II / Pyruvate:ferredoxin oxidoreductase core domain II / Pyruvate flavodoxin/ferredoxin oxidoreductase, pyrimidine binding domain / : / Pyruvate flavodoxin/ferredoxin oxidoreductase, thiamine diP-bdg / Pyruvate-flavodoxin oxidoreductase, central domain / Pyruvate/ketoisovalerate oxidoreductase, catalytic domain / Pyruvate ferredoxin/flavodoxin oxidoreductase / Rossmann fold - #920 / Transketolase C-terminal/Pyruvate-ferredoxin oxidoreductase domain II / Thiamine pyrophosphate enzyme, C-terminal TPP-binding / Thiamine pyrophosphate enzyme, C-terminal TPP binding domain / Thiamin diphosphate (ThDP)-binding fold, Pyr/PP domains / Thiamin diphosphate-binding fold / 4Fe-4S dicluster domain / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / THIAMINE DIPHOSPHATE / Pyruvate:ferredoxin oxidoreductase
Similarity search - Component
Biological speciesDesulfocurvibacter africanus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsCherrier, M.V. / Vernede, X. / Fenel, D. / Martin, L. / Arragain, B. / Neumann, E. / Fontecilla Camps, J.C. / Schoehn, G. / Nicolet, Y.
Funding support France, 4items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-15-IDEX-02 France
Grenoble Instruct-ERIC Center (ISBG)UMS 3518 CNRS-CEA-UGA-EMBL France
French Infrastructure for Integrated Structural Biology (FRISBI)ANR-10-INSB-05-02 France
Grenoble Alliance for Integrated Structural Cell Biology (GRAL)ANR-10-LABX-49-01 France
CitationJournal: Biomolecules / Year: 2022
Title: Oxygen-Sensitive Metalloprotein Structure Determination by Cryo-Electron Microscopy.
Authors: Mickaël V Cherrier / Xavier Vernède / Daphna Fenel / Lydie Martin / Benoit Arragain / Emmanuelle Neumann / Juan C Fontecilla-Camps / Guy Schoehn / Yvain Nicolet /
Abstract: Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] ...Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [FeS]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.
History
DepositionAug 31, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 23, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pyruvate:ferredoxin oxidoreductase
B: Pyruvate:ferredoxin oxidoreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,75914
Polymers267,6702
Non-polymers3,08912
Water2,054114
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area30120 Å2
ΔGint-294 kcal/mol
Surface area70780 Å2
MethodPISA

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Pyruvate:ferredoxin oxidoreductase / PFOR / POR / Pyruvate synthase


Mass: 133835.109 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfocurvibacter africanus (bacteria)
Gene: por / Production host: Desulfocurvibacter africanus (bacteria) / References: UniProt: P94692, pyruvate synthase

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Non-polymers , 5 types, 126 molecules

#2: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Fe4S4
#3: Chemical ChemComp-TPP / THIAMINE DIPHOSPHATE


Mass: 425.314 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C12H19N4O7P2S / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 114 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: PYRUVATE FERREDOXIN OXIDOREDUCTASE / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.267360 MDa / Experimental value: NO
Source (natural)Organism: Desulfocurvibacter africanus (bacteria)
Buffer solutionpH: 8.4
Buffer componentConc.: 10 mM / Name: Tris
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: PROPANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1304
Image scansMovie frames/image: 60 / Used frames/image: 2-60

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Processing

SoftwareName: PHENIX / Version: (1.20.1_4487:phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4.1CTF correction
7PHENIXmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180868 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: RECIPROCAL
Atomic model buildingPDB-ID: 1B0P

1b0p


Pdb chain-ID: A / Accession code: 1B0P / Source name: PDB / Type: experimental model
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 100.48 Å2 / Biso mean: 32.3636 Å2 / Biso min: 0 Å2

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