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- EMDB-25397: CSDaV GFP mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-25397
TitleCSDaV GFP mutant
Map data
Sample
  • Virus: Citrus sudden death-associated virus
    • Protein or peptide: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
Function / homology
Function and homology information


mRNA methyltransferase activity / RNA processing / bioluminescence / generation of precursor metabolites and energy / viral capsid / RNA helicase activity / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription ...mRNA methyltransferase activity / RNA processing / bioluminescence / generation of precursor metabolites and energy / viral capsid / RNA helicase activity / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / structural molecule activity / proteolysis / RNA binding / ATP binding
Similarity search - Function
Peptidase C21 / Tymovirus endopeptidase domain / Salyut domain / Tymovirus endopeptidase / Salyut domain / Tymovirus coat protein / Tymovirus coat protein / Viral methyltransferase / Alphavirus-like methyltransferase (MT) domain / Alphavirus-like methyltransferase (MT) domain profile. ...Peptidase C21 / Tymovirus endopeptidase domain / Salyut domain / Tymovirus endopeptidase / Salyut domain / Tymovirus coat protein / Tymovirus coat protein / Viral methyltransferase / Alphavirus-like methyltransferase (MT) domain / Alphavirus-like methyltransferase (MT) domain profile. / Tymovirus, RNA-dependent RNA polymerase / RNA dependent RNA polymerase / Viral (Superfamily 1) RNA helicase / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Viral coat protein subunit / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Green fluorescent protein / Polyprotein
Similarity search - Component
Biological speciesCitrus sudden death-associated virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGuo F / Matsumura EE / Falk BW
Funding support United States, 1 items
OrganizationGrant numberCountry
United States Department of Agriculture (USDA) United States
CitationJournal: Biotechnol Rep (Amst) / Year: 2022
Title: Citrus sudden death-associated virus as a new expression vector for rapid production of heterologous proteins, chimeric virions, and virus-like particles.
Authors: Emilyn E Matsumura / Fei Guo / Daan Boogers / Dennis van Oevelen / Sandra T Vu / Bryce W Falk /
Abstract: The more we understand the strategies used by viruses for protein expression, the more possibilities we have to exploit viruses as expression vectors for heterologous protein production. Advances in ...The more we understand the strategies used by viruses for protein expression, the more possibilities we have to exploit viruses as expression vectors for heterologous protein production. Advances in the development of virus-based expression systems have been possible due to generation of many virus infectious clones, especially those derived from plant viruses, which have the capability for rapid and high-level transient expression of proteins in plant cells, a robust and low-cost bioreactor. In this work, we generated new replicative virus expression vectors based on a previously constructed citrus sudden death-associated virus (CSDaV) infectious cDNA clone. These vectors were generated to express the reporter green fluorescent protein (GFP) in leaves by taking advantage of the expression strategies used by CSDaV to produce its structural proteins. We show that higher amounts of GFP can be produced from a coat protein (CP)-independent CSDaV-based vector, compared to levels of GFP expressed from a widely used non-replicative vector (pEAQ series); or GFP can be produced in fusion with the major CSDaV CP (CPp21) to be incorporated into chimeric virions. However, GFP-recombinant CSDaV virions do not appear uniformly assembled, but more likely as mosaic particles. Cryo-electron microscopy analysis from this work revealed the structures of the wild-type and the GFP-recombinant CSDaV virions, but it was not able to reveal where exactly the GFP is displayed in the chimeric virions. We show though that the incorporation of GFP-CPp21 fusion protein into virions occurs solely due to its interaction with free/non-fused CPp21, independent of other viral proteins. Therefore, individual co-expression of GFP-CPp21 and CPp21 in the same plant cells leads to the production of chimeric virus-like particles (VLPs), while GFP-CPp21 fusion protein itself is not able to self-assemble into VLPs. The new CSDaV-based expression vectors may provide an alternative platform for use in molecular farming, either for production of heterologous proteins or as scaffold for heterologous protein display.
History
DepositionNov 7, 2021-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25397.png

Downloads & links

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Map

FileDownload / File: emd_25397.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.022450013 - 0.0695851
Average (Standard dev.)0.0014562452 (±0.004621018)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 450.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Citrus sudden death-associated virus

EntireName: Citrus sudden death-associated virus
Components
  • Virus: Citrus sudden death-associated virus
    • Protein or peptide: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein

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Supramolecule #1: Citrus sudden death-associated virus

SupramoleculeName: Citrus sudden death-associated virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: GFP is linked at the N-terminus of each capsid protein.
Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No

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Macromolecule #1: Citrus Sudden Death-associated Virus Capsid Protein,Green fluores...

MacromoleculeName: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
type: protein_or_peptide / ID: 1
Details: N-terminus MQSDTLLP is from p25, GFP is sandwiched by two viral protease cleavage sites LTGG and LTGGFS, which is followed by p21
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Citrus sudden death-associated virus
Molecular weightTheoretical: 49.358574 KDa
Recombinant expressionOrganism: Agrobacterium tumefaciens (bacteria)
SequenceString: MQSDTLLPLT GGVSKGEELF TGVVPILVEL DGDVNGHKFS VSGEGEGDAT YGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDH MKQHDFFKSA MPEGYVQERT IFFKDDGNYK TRAEVKFEGD TLVNRIELKG IDFKEDGNIL GHKLEYNYNS H NVYIMADK ...String:
MQSDTLLPLT GGVSKGEELF TGVVPILVEL DGDVNGHKFS VSGEGEGDAT YGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDH MKQHDFFKSA MPEGYVQERT IFFKDDGNYK TRAEVKFEGD TLVNRIELKG IDFKEDGNIL GHKLEYNYNS H NVYIMADK QKNGIKVNFK IRHNIEDGSV QLADHYQQNT PIGDGPVLLP DNHYLSTQSA LSKDPNEKRD HMVLLEFVTA AG ITLGMDE LYKLTGGFSM ASDAQAGPAP SRDDRVDRQP RLPAAPRVAE VGLNAPSVDY PFQWVVASYD GSEAKNLSDD LSG SATLTK VMANYRHAEL TSVELEVCPL AAAFSKPISV SAVWTIASIS PASASETSYY GGRLFTVGGP VLMSSTTHLP ADLT RLNPV LKGPVKYTDC PRFSYSVYSN GGTKGTNLCT IILRGVVRLS GPSGNLLA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III
Details: blot for 8 seconds before plunging with -2mm off set.

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 56818 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 90.0 K
DetailsDirect alignment is done from microscope side, then COMA free alignment and CTF based astigmatism correction is done using SerialEM.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7890 / Average exposure time: 3.0 sec. / Average electron dose: 60.0 e/Å2

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Image processing

Particle selectionNumber selected: 60337 / Details: LoG based particle selection using Relion 3.1
CTF correctionSoftware - Name: RELION (ver. 3.1) / Software - details: CTffind4
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 5600 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Details: Relion is used for finial reconstruction / Number images used: 12732
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsAb initio model of an ASU is built in Coot and the corresponding density map is then cut out using UCSF Chimera. The model is then refined in Phenix.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7sqy:
CSDaV GFP mutant

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