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- PDB-7sqy: CSDaV GFP mutant -

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Basic information

Entry
Database: PDB / ID: 7sqy
TitleCSDaV GFP mutant
ComponentsCitrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
KeywordsVIRUS / Capsid / coat protein
Function / homology
Function and homology information


mRNA methyltransferase activity / mRNA modification / 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 ...mRNA methyltransferase activity / mRNA modification / 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 / Peptidase family C21 domain profile. / Tymovirus coat protein / Tymovirus coat protein / Viral methyltransferase / Alphavirus-like methyltransferase (MT) domain ...Peptidase C21 / Tymovirus endopeptidase domain / Salyut domain / Tymovirus endopeptidase / Salyut domain / Peptidase family C21 domain profile. / 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
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGuo, F. / Matsumura, E.E. / Falk, B.W.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 25, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 22, 2022Group: Database references / Category: citation / Item: _citation.page_first / _citation.page_last
Revision 1.3Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
B: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
C: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein


Theoretical massNumber of molelcules
Total (without water)148,0763
Polymers148,0763
Non-polymers00
Water00
1
A: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
B: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
C: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
x 60


Theoretical massNumber of molelcules
Total (without water)8,884,543180
Polymers8,884,543180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
B: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
C: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
x 5


  • icosahedral pentamer
  • 740 kDa, 15 polymers
Theoretical massNumber of molelcules
Total (without water)740,37915
Polymers740,37915
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
B: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
C: Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein
x 6


  • icosahedral 23 hexamer
  • 888 kDa, 18 polymers
Theoretical massNumber of molelcules
Total (without water)888,45418
Polymers888,45418
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Citrus Sudden Death-associated Virus Capsid Protein,Green fluorescent protein,Citrus Sudden Death-associated Virus Capsid Protein


Mass: 49358.574 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: N-terminus MQSDTLLP is from p25, GFP is sandwiched by two viral protease cleavage sites LTGG and LTGGFS, which is followed by p21
Source: (gene. exp.) Citrus sudden death-associated virus, (gene. exp.) Aequorea victoria (jellyfish)
Gene: GFP / Production host: Agrobacterium tumefaciens (bacteria) / References: UniProt: Q3HWZ1, UniProt: P42212

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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: Citrus sudden death-associated virus / Type: VIRUS
Details: GFP is linked at the N-terminus of each capsid protein.
Entity ID: all / Source: MULTIPLE SOURCES
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K
Details: blot for 8 seconds before plunging with -2mm off set

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Details: Direct alignment is done from microscope side, then COMA free alignment and CTF based astigmatism correction is done using SerialEM.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Calibrated magnification: 56818 X / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 90 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7890

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selection
2SerialEMimage acquisition
4RELION3.1CTF correctionCTffind4
7UCSF Chimeramodel fitting
9PHENIX1.19.2model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 60337 / Details: LoG based particle selection using Relion 3.1
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12732 / Algorithm: BACK PROJECTION / Details: Relion is used for finial reconstruction / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: Ab 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.
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 23.33 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00343951
ELECTRON MICROSCOPYf_angle_d0.55015405
ELECTRON MICROSCOPYf_chiral_restr0.0443640
ELECTRON MICROSCOPYf_plane_restr0.0032687
ELECTRON MICROSCOPYf_dihedral_angle_d4.058565

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