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- PDB-8orh: Knockout of GMC-oxidoreductase genes reveals that functional redu... -

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Basic information

Entry
Database: PDB / ID: 8orh
TitleKnockout of GMC-oxidoreductase genes reveals that functional redundancy preserves mimivirus essential functions
ComponentsPutative GMC-type oxidoreductase
KeywordsSTRUCTURAL PROTEIN / GMC-oxidoreductase / genomic fiber / mimivirus
Function / homology
Function and homology information


choline dehydrogenase activity / glycine betaine biosynthetic process from choline / flavin adenine dinucleotide binding
Similarity search - Function
GMC oxidoreductases signature 2. / Glucose-methanol-choline oxidoreductase / Glucose-methanol-choline oxidoreductase, N-terminal / GMC oxidoreductase / Glucose-methanol-choline oxidoreductase, C-terminal / GMC oxidoreductase / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / GMC-type oxidoreductase
Similarity search - Component
Biological speciesMimivirus reunion
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsAlempic, J.M. / Bisio, H. / Villalta, A. / Santini, S. / Lartigue, A. / Schmitt, A. / Bugnot, C. / Notaro, A. / Belmudes, L. / Adrait, A. ...Alempic, J.M. / Bisio, H. / Villalta, A. / Santini, S. / Lartigue, A. / Schmitt, A. / Bugnot, C. / Notaro, A. / Belmudes, L. / Adrait, A. / Poirot, O. / Ptchelkine, D. / De Castro, C. / Coute, Y. / Abergel, C.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)832601European Union
CitationJournal: Microlife / Year: 2024
Title: Functional redundancy revealed by the deletion of the mimivirus GMC-oxidoreductase genes.
Authors: Jean-Marie Alempic / Hugo Bisio / Alejandro Villalta / Sébastien Santini / Audrey Lartigue / Alain Schmitt / Claire Bugnot / Anna Notaro / Lucid Belmudes / Annie Adrait / Olivier Poirot / ...Authors: Jean-Marie Alempic / Hugo Bisio / Alejandro Villalta / Sébastien Santini / Audrey Lartigue / Alain Schmitt / Claire Bugnot / Anna Notaro / Lucid Belmudes / Annie Adrait / Olivier Poirot / Denis Ptchelkine / Cristina De Castro / Yohann Couté / Chantal Abergel /
Abstract: The mimivirus 1.2 Mb genome was shown to be organized into a nucleocapsid-like genomic fiber encased in the nucleoid compartment inside the icosahedral capsid. The genomic fiber protein shell is ...The mimivirus 1.2 Mb genome was shown to be organized into a nucleocapsid-like genomic fiber encased in the nucleoid compartment inside the icosahedral capsid. The genomic fiber protein shell is composed of a mixture of two GMC-oxidoreductase paralogs, one of them being the main component of the glycosylated layer of fibrils at the surface of the virion. In this study, we determined the effect of the deletion of each of the corresponding genes on the genomic fiber and the layer of surface fibrils. First, we deleted the GMC-oxidoreductase, the most abundant in the genomic fiber, and determined its structure and composition in the mutant. As expected, it was composed of the second GMC-oxidoreductase and contained 5- and 6-start helices similar to the wild-type fiber. This result led us to propose a model explaining their coexistence. Then we deleted the GMC-oxidoreductase, the most abundant in the layer of fibrils, to analyze its protein composition in the mutant. Second, we showed that the fitness of single mutants and the double mutant were not decreased compared with the wild-type viruses under laboratory conditions. Third, we determined that deleting the GMC-oxidoreductase genes did not impact the glycosylation or the glycan composition of the layer of surface fibrils, despite modifying their protein composition. Because the glycosylation machinery and glycan composition of members of different clades are different, we expanded the analysis of the protein composition of the layer of fibrils to members of the B and C clades and showed that it was different among the three clades and even among isolates within the same clade. Taken together, the results obtained on two distinct central processes (genome packaging and virion coating) illustrate an unexpected functional redundancy in members of the family , suggesting this may be the major evolutionary force behind their giant genomes.
History
DepositionApr 14, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_id_ISSN ..._citation.country / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putative GMC-type oxidoreductase
B: Putative GMC-type oxidoreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,6074
Polymers154,0362
Non-polymers1,5712
Water00
1


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

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Components

#1: Protein Putative GMC-type oxidoreductase


Mass: 77018.023 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Details: soruce organism: mimivirus reunion mutant where the qu_946 gene has been replaced by a selection cassette.
Source: (natural) Mimivirus reunion / Variant: KO_qu946 / Strain: HB1931 / References: UniProt: A0A8A5IZP6
#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Mimivirus reunion / Type: VIRUS
Details: in the source organism, qu_946 gene has been removed and replaced by a selection cassette.
Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Mimivirus reunion
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Acanthamoeba castellanii str. Neff / Strain: KO_qu946
Virus shellName: genomic fiber / Diameter: 320 nm
Buffer solutionpH: 7.5
Buffer componentConc.: 40 mmol/l / Name: Tris-HCl / Formula: Tris-HCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: genomic fiber purified on ClCs gradient
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.3 sec. / Electron dose: 35.597 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 2224
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selection
2EPU2.11.1image acquisition
4CTFFIND4.1.5CTF correction
7PHENIX1.20.1model fitting
11RELION3.1classification
12RELION43D reconstructionpolishing+refine with solvent flattening
13PHENIX1.20.1model refinementafter manual refinement in coot
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 49.426 ° / Axial rise/subunit: 20.497 Å / Axial symmetry: C3
Particle selectionNum. of particles selected: 172431
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 20899 / Num. of class averages: 2 / Symmetry type: HELICAL
Atomic model buildingB value: 40.56 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 4Z24

4z24


Accession code: 4Z24 / Chain residue range: 4-652 / Pdb chain residue range: 4-652 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310380
ELECTRON MICROSCOPYf_angle_d0.50114178
ELECTRON MICROSCOPYf_dihedral_angle_d7.2281428
ELECTRON MICROSCOPYf_chiral_restr0.0461572
ELECTRON MICROSCOPYf_plane_restr0.0051844

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