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- PDB-7lcg: The mature Usutu SAAR-1776, Model A -

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Basic information

Entry
Database: PDB / ID: 7lcg
TitleThe mature Usutu SAAR-1776, Model A
Components
  • Envelope protein E
  • Membrane protein MBiological membrane
KeywordsVIRUS / Flavivirus / envelope glycoprotein
Function / homology
Function and homology information


symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / ribonucleoside triphosphate phosphatase activity / viral capsid / double-stranded RNA binding / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / RNA helicase activity / membrane => GO:0016020 / host cell endoplasmic reticulum membrane / protein dimerization activity ...symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / ribonucleoside triphosphate phosphatase activity / viral capsid / double-stranded RNA binding / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / RNA helicase activity / membrane => GO:0016020 / host cell endoplasmic reticulum membrane / protein dimerization activity / symbiont entry into host cell / viral RNA genome replication / RNA-dependent RNA polymerase activity / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell nucleus / virion attachment to host cell / virion membrane / structural molecule activity / proteolysis / extracellular region / ATP binding / metal ion binding
Similarity search - Function
Flavivirus capsid protein C superfamily / Flavivirus non-structural protein NS2B / Genome polyprotein, Flavivirus / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B / mRNA cap 0/1 methyltransferase / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B ...Flavivirus capsid protein C superfamily / Flavivirus non-structural protein NS2B / Genome polyprotein, Flavivirus / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B / mRNA cap 0/1 methyltransferase / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B / Flavivirus non-structural protein NS4A / Flavivirus NS2B domain profile. / mRNA cap 0 and cap 1 methyltransferase (EC 2.1.1.56 and EC 2.1.1.57) domain profile. / Flavivirus non-structural protein NS2A / Flavivirus non-structural protein NS2A / Flavivirus NS3, petidase S7 / Peptidase S7, Flavivirus NS3 serine protease / Flavivirus NS3 protease (NS3pro) domain profile. / Envelope glycoprotein M, flavivirus / Flavivirus envelope glycoprotein M / RNA-directed RNA polymerase, flavivirus / Flavivirus RNA-directed RNA polymerase, fingers and palm domains / Flavivirus non-structural Protein NS1 / Flavivirus non-structural protein NS1 / Envelope glycoprotein M superfamily, flavivirus / Flavivirus polyprotein propeptide / Flavivirus polyprotein propeptide superfamily / Flavivirus polyprotein propeptide / Flaviviral glycoprotein E, central domain, subdomain 1 / Flaviviral glycoprotein E, central domain, subdomain 2 / Flavivirus envelope glycoprotein E, Stem/Anchor domain / Flavivirus glycoprotein E, immunoglobulin-like domain / Flavivirus envelope glycoprotein E, Stem/Anchor domain superfamily / Flavivirus glycoprotein, immunoglobulin-like domain / Flavivirus glycoprotein central and dimerisation domain / Flavivirus glycoprotein, central and dimerisation domains / Ribosomal RNA methyltransferase, FtsJ domain / FtsJ-like methyltransferase / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / Flavivirus glycoprotein, central and dimerisation domain superfamily / Flaviviral glycoprotein E, dimerisation domain / DEAD box, Flavivirus / Flavivirus DEAD domain / helicase superfamily c-terminal domain / Immunoglobulin E-set / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-0SM / Chem-PC7 / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesUsutu virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.42 Å
AuthorsKhare, B. / Klose, T. / Fang, Q. / Kuhn, R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI076331 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI095366 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry.
Authors: Baldeep Khare / Thomas Klose / Qianglin Fang / Michael G Rossmann / Richard J Kuhn /
Abstract: Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive ...Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.
History
DepositionJan 11, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Envelope protein E
B: Membrane protein M
C: Envelope protein E
D: Membrane protein M
E: Envelope protein E
F: Membrane protein M
hetero molecules


Theoretical massNumber of molelcules
Total (without water)191,10218
Polymers185,8736
Non-polymers5,23012
Water0
1
A: Envelope protein E
B: Membrane protein M
C: Envelope protein E
D: Membrane protein M
E: Envelope protein E
F: Membrane protein M
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)11,466,1391080
Polymers11,152,354360
Non-polymers313,785720
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: Envelope protein E
B: Membrane protein M
C: Envelope protein E
D: Membrane protein M
E: Envelope protein E
F: Membrane protein M
hetero molecules
x 5


  • icosahedral pentamer
  • 956 kDa, 30 polymers
Theoretical massNumber of molelcules
Total (without water)955,51290
Polymers929,36330
Non-polymers26,14960
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: Envelope protein E
B: Membrane protein M
C: Envelope protein E
D: Membrane protein M
E: Envelope protein E
F: Membrane protein M
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 1.15 MDa, 36 polymers
Theoretical massNumber of molelcules
Total (without water)1,146,614108
Polymers1,115,23536
Non-polymers31,37872
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
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 Envelope protein E /


Mass: 53623.812 Da / Num. of mol.: 3 / Fragment: UNP residues 294-793
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Usutu virus / Strain: SAAR-1776 / Cell line (production host): Vero / Production host: Chlorocebus aethiops (grivet) / References: UniProt: Q5WPU4
#2: Protein Membrane protein M / Biological membrane


Mass: 8333.710 Da / Num. of mol.: 3 / Fragment: UNP residues 219-293
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Usutu virus / Strain: SAAR-1776 / Cell line (production host): Vero / Production host: Chlorocebus aethiops (grivet) / References: UniProt: A0A0H3U5P6
#3: Chemical ChemComp-0SM / TRIMETHYL-[2-[[(2S,3S)-2-(OCTADECANOYLAMINO)-3-OXIDANYL-BUTOXY]-OXIDANYL-PHOSPHORYL]OXYETHYL]AZANIUM / N-OCTADECANOYL-D-ERYTHRO-SPHINGOSYLPHOSPHORYLCHOLINE


Mass: 537.733 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H58N2O6P / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-PC7 / (7S)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / PHOSPHATIDYLCHOLINE / 1-PALMITOYL-2-STEAROYL-PC / Phosphatidylcholine


Mass: 763.100 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C42H85NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY

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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: Usutu virus / Type: VIRUS / Details: Virus purified using Vero cells. / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Usutu virus / Strain: SAAR-1776
Source (recombinant)Organism: Chlorocebus aethiops (grivet) / Cell: Vero cells
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Turdus merula
Buffer solutionpH: 8 / Details: 20 mM Tris, 120 mM NaCl, 1 mM EDTA, pH 8.0
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisNH2C(CH2OH)31
2120 mMSodium chlorideNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acid(HO2CCH2)2NCH2CH2N(CH2CO2H)21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.08 sec. / Electron dose: 23.68 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3689
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.19rc5_4047: / Classification: refinement
EM software
IDNameCategory
2Leginonimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10jsprinitial Euler assignment
11jsprfinal Euler assignment
12jsprclassification
13jspr3D reconstruction
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101311 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 5WSN

5wsn

RefinementHighest resolution: 2.42 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00513315
ELECTRON MICROSCOPYf_angle_d0.62718060
ELECTRON MICROSCOPYf_dihedral_angle_d5.3481843
ELECTRON MICROSCOPYf_chiral_restr0.0472067
ELECTRON MICROSCOPYf_plane_restr0.0052286

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