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- EMDB-17131: Knockout of GMC-oxidoreductase genes reveals that functional redu... -

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Basic information

Entry
Database: EMDB / ID: EMD-17131
TitleKnockout of GMC-oxidoreductase genes reveals that functional redundancy preserves mimivirus essential functions
Map dataCL1 masked post-processed main map
Sample
  • Virus: Mimivirus reunion
    • Protein or peptide: Putative GMC-type oxidoreductase
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
KeywordsGMC-oxidoreductase / genomic fiber / mimivirus / STRUCTURAL PROTEIN
Function / homology
Function and homology information


oxidoreductase activity, acting on CH-OH group of donors / 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
GMC-type oxidoreductase
Similarity search - Component
Biological speciesMimivirus reunion
Methodhelical reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsAlempic JM / Bisio H / Villalta A / Santini S / Lartigue A / Schmitt A / Bugnot C / Notaro A / Belmudes L / Adrait A ...Alempic JM / 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, 1 items
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 17, 2023-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17131.png

Downloads & links

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Map

FileDownload / File: emd_17131.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCL1 masked post-processed main map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 400 pix.
= 434.36 Å		1.09 Å/pix.
x 400 pix.
= 434.36 Å		1.09 Å/pix.
x 400 pix.
= 434.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0859 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.0144134 - 0.042150997
Average (Standard dev.)0.00066858676 (±0.0025330277)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 434.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: CL1 half map 1

Fileemd_17131_half_map_1.map
AnnotationCL1 half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CL1 half map 2

Fileemd_17131_half_map_2.map
AnnotationCL1 half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mimivirus reunion

EntireName: Mimivirus reunion
Components
  • Virus: Mimivirus reunion
    • Protein or peptide: Putative GMC-type oxidoreductase
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE

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Supramolecule #1: Mimivirus reunion

SupramoleculeName: Mimivirus reunion / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: in the source organism, qu_946 gene has been removed and replaced by a selection cassette.
NCBI-ID: 2813486 / Sci species name: Mimivirus reunion / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Acanthamoeba castellanii str. Neff (eukaryote) / Strain: KO_qu946
Virus shellShell ID: 1 / Name: genomic fiber / Diameter: 300.0 Å

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Macromolecule #1: Putative GMC-type oxidoreductase

MacromoleculeName: Putative GMC-type oxidoreductase / type: protein_or_peptide / ID: 1
Details: source organism: mimivirus reunion mutant where the qu_946 gene has been replaced by a selection cassette.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mimivirus reunion / Strain: HB1931
Molecular weightTheoretical: 77.018023 KDa
SequenceString: MKNRECCKCY NPCEKICVNY STTDVAFERP NPCKPTPCKP TPIPCDPCHN TKDNLTGDIV IIGAGAAGSL LAHYLARFSN MKIILLEAG HSHFNDPVVT DPMGFFGKYN PPNENISMSQ NPSYSWQGAQ EPNTGAYGNR PIIAHGMGFG GSTMINRLNL V VGGRTVFD ...String:
MKNRECCKCY NPCEKICVNY STTDVAFERP NPCKPTPCKP TPIPCDPCHN TKDNLTGDIV IIGAGAAGSL LAHYLARFSN MKIILLEAG HSHFNDPVVT DPMGFFGKYN PPNENISMSQ NPSYSWQGAQ EPNTGAYGNR PIIAHGMGFG GSTMINRLNL V VGGRTVFD NDWPVGWKYD DVKNYFRRVL VDINPVRDNT KASITSVALD ALRIIAEQQI ASGEPVDFLL NKATGNVPNV EK TTPDAVP LNLNDYEGVN SVVAFSSFYM GVNQLSDGNY IRKYAGNTYL NRNYVDENGR GIGKFSGLRV VSDAVVDRII FKG NRAVGV NYIDREGIMH YVKVNKEVVV TSGAFYTPTI LQRSGIGDFT YLSSIGVKNL VYNNPLVGTG LKNHYSPVTI TRVH GEPSE VSRFLSNMAA NPTNMGFKGL AELGFHRLDP NKPANANTVT YRKYQLMMTA GVGIPAEQQY LSGLSPSSNN LFTLI ADDI RFAPEGYIKI GTPNIPRDVP KIFFNTFVTY TPTSAPADQQ WPIAQKTLAP LISALLGYDI IYQTLISMNQ TARDSG FQV SLEMVYPLND LIYKLHNGLA TYGANWWHYF VPTLVGDDTP AGREFADTLS KLSYYPRVGA HLDSHQGCSC SIGRTVD SN LKVIGTQNVR VADLSAAAFP PGGNTWATAS MIGARAVDLI LGFPYLRDLP VNDVPILNVN

UniProtKB: GMC-type oxidoreductase

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Macromolecule #2: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 2 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5 / Component - Concentration: 40.0 mmol/l / Component - Formula: Tris-HCl / Component - Name: Tris-HCl
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailsgenomic fiber purified on ClCs gradient

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 2224 / Average exposure time: 2.3 sec. / Average electron dose: 35.597 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 2
Applied symmetry - Helical parameters - Δz: 7.949 Å
Applied symmetry - Helical parameters - Δ&Phi: 138.921 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 4.0)
Software - details: polishing+refine with solvent flattening
Number images used: 98882
Segment selectionNumber selected: 442237 / Software - Name: RELION (ver. 3.1)
Startup modelType of model: OTHER / Details: 300 A diameter reference cylindre.
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4z24


Chain - Residue range: 4-652 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 86.35
Output model

PDB-8ors:
Knockout of GMC-oxidoreductase genes reveals that functional redundancy preserves mimivirus essential functions

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