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- PDB-6b43: CryoEM structure and atomic model of the Kaposi's sarcoma-associa... -

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Basic information

Entry
Database: PDB / ID: 6b43
TitleCryoEM structure and atomic model of the Kaposi's sarcoma-associated herpesvirus capsid
Components
  • Major capsid protein
  • Small capsomere-interacting protein
  • Triplex capsid protein 1
  • Triplex capsid protein 2
KeywordsVIRUS / human herpesvirus 8 / human tumor virus / dsDNA virus capsid assembly / HK97-like fold
Function / homology
Function and homology information


T=16 icosahedral viral capsid / viral capsid assembly / viral process / virion component / viral capsid / host cell nucleus / structural molecule activity / DNA binding
Similarity search - Function
Gammaherpesvirus capsid / Gammaherpesvirus capsid protein / Herpesvirus capsid shell protein 1 / Herpesvirus capsid shell protein VP19C / Herpesvirus capsid protein 2 / Herpesvirus VP23 like capsid protein / Herpesvirus major capsid protein / Herpesvirus major capsid protein, upper domain superfamily / Herpes virus major capsid protein
Similarity search - Domain/homology
ORF26 / ORF25 / Small capsomere-interacting protein / Core gene UL38 family protein
Similarity search - Component
Biological speciesHuman herpesvirus 8
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsDai, X.H. / Gong, D.Y. / Sun, R. / Zhou, Z.H.
Funding support United States, 8items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)DE025567 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA091791 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA177322 United States
National Institutes of Health/Office of the Director1S10OD018111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1U24GM116792 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
CitationJournal: Nature / Year: 2018
Title: Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication.
Authors: Xinghong Dai / Danyang Gong / Hanyoung Lim / Jonathan Jih / Ting-Ting Wu / Ren Sun / Z Hong Zhou /
Abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer that commonly affects patients with AIDS and which is endemic in sub-Saharan Africa. The KSHV capsid is highly ...Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer that commonly affects patients with AIDS and which is endemic in sub-Saharan Africa. The KSHV capsid is highly pressurized by its double-stranded DNA genome, as are the capsids of the eight other human herpesviruses. Capsid assembly and genome packaging of herpesviruses are prone to interruption and can therefore be targeted for the structure-guided development of antiviral agents. However, herpesvirus capsids-comprising nearly 3,000 proteins and over 1,300 Å in diameter-present a formidable challenge to atomic structure determination and functional mapping of molecular interactions. Here we report a 4.2 Å resolution structure of the KSHV capsid, determined by electron-counting cryo-electron microscopy, and its atomic model, which contains 46 unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP) and the triplex proteins Tri1 and Tri2. Our structure and mutagenesis results reveal a groove in the upper domain of the MCP that contains hydrophobic residues that interact with the SCP, which in turn crosslinks with neighbouring MCPs in the same hexon to stabilize the capsid. Multiple levels of MCP-MCP interaction-including six sets of stacked hairpins lining the hexon channel, disulfide bonds across channel and buttress domains in neighbouring MCPs, and an interaction network forged by the N-lasso domain and secured by the dimerization domain-define a robust capsid that is resistant to the pressure exerted by the enclosed genome. The triplexes, each composed of two Tri2 molecules and a Tri1 molecule, anchor to the capsid floor via a Tri1 N-anchor to plug holes in the MCP network and rivet the capsid floor. These essential roles of the MCP N-lasso and Tri1 N-anchor are verified by serial-truncation mutageneses. Our proof-of-concept demonstration of the use of polypeptides that mimic the smallest capsid protein to inhibit KSHV lytic replication highlights the potential for exploiting the interaction hotspots revealed in our atomic structure to develop antiviral agents.
History
DepositionSep 25, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 8, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2017Group: Author supporting evidence / Other / Category: cell / pdbx_audit_support
Item: _cell.Z_PDB / _cell.length_a ..._cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c / _pdbx_audit_support.funding_organization
Revision 1.2Jan 31, 2018Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.3Feb 7, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
SupersessionApr 11, 2018ID: 3J9A
Revision 1.4Apr 11, 2018Group: Advisory / Data collection / Category: pdbx_database_PDB_obs_spr
Revision 1.5Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.7Nov 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

Movie
  • 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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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Major capsid protein
B: Major capsid protein
C: Major capsid protein
D: Major capsid protein
E: Major capsid protein
F: Major capsid protein
G: Small capsomere-interacting protein
H: Small capsomere-interacting protein
I: Small capsomere-interacting protein
J: Small capsomere-interacting protein
K: Small capsomere-interacting protein
L: Small capsomere-interacting protein
M: Major capsid protein
N: Major capsid protein
O: Major capsid protein
P: Small capsomere-interacting protein
Q: Small capsomere-interacting protein
R: Small capsomere-interacting protein
S: Major capsid protein
T: Major capsid protein
U: Major capsid protein
V: Major capsid protein
W: Major capsid protein
X: Major capsid protein
Y: Small capsomere-interacting protein
Z: Small capsomere-interacting protein
0: Small capsomere-interacting protein
1: Small capsomere-interacting protein
2: Small capsomere-interacting protein
3: Small capsomere-interacting protein
4: Major capsid protein
5: Triplex capsid protein 1
6: Triplex capsid protein 2
7: Triplex capsid protein 2
8: Triplex capsid protein 1
9: Triplex capsid protein 2
a: Triplex capsid protein 2
b: Triplex capsid protein 1
c: Triplex capsid protein 2
d: Triplex capsid protein 2
e: Triplex capsid protein 1
f: Triplex capsid protein 2
g: Triplex capsid protein 2
h: Triplex capsid protein 1
i: Triplex capsid protein 2
j: Triplex capsid protein 2


Theoretical massNumber of molelcules
Total (without water)3,260,81346
Polymers3,260,81346
Non-polymers00
Water00
1
A: Major capsid protein
B: Major capsid protein
C: Major capsid protein
D: Major capsid protein
E: Major capsid protein
F: Major capsid protein
G: Small capsomere-interacting protein
H: Small capsomere-interacting protein
I: Small capsomere-interacting protein
J: Small capsomere-interacting protein
K: Small capsomere-interacting protein
L: Small capsomere-interacting protein
M: Major capsid protein
N: Major capsid protein
O: Major capsid protein
P: Small capsomere-interacting protein
Q: Small capsomere-interacting protein
R: Small capsomere-interacting protein
S: Major capsid protein
T: Major capsid protein
U: Major capsid protein
V: Major capsid protein
W: Major capsid protein
X: Major capsid protein
Y: Small capsomere-interacting protein
Z: Small capsomere-interacting protein
0: Small capsomere-interacting protein
1: Small capsomere-interacting protein
2: Small capsomere-interacting protein
3: Small capsomere-interacting protein
4: Major capsid protein
5: Triplex capsid protein 1
6: Triplex capsid protein 2
7: Triplex capsid protein 2
8: Triplex capsid protein 1
9: Triplex capsid protein 2
a: Triplex capsid protein 2
b: Triplex capsid protein 1
c: Triplex capsid protein 2
d: Triplex capsid protein 2
e: Triplex capsid protein 1
f: Triplex capsid protein 2
g: Triplex capsid protein 2
h: Triplex capsid protein 1
i: Triplex capsid protein 2
j: Triplex capsid protein 2
x 60


Theoretical massNumber of molelcules
Total (without water)195,648,7982760
Polymers195,648,7982760
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: Major capsid protein
B: Major capsid protein
C: Major capsid protein
D: Major capsid protein
E: Major capsid protein
F: Major capsid protein
G: Small capsomere-interacting protein
H: Small capsomere-interacting protein
I: Small capsomere-interacting protein
J: Small capsomere-interacting protein
K: Small capsomere-interacting protein
L: Small capsomere-interacting protein
M: Major capsid protein
N: Major capsid protein
O: Major capsid protein
P: Small capsomere-interacting protein
Q: Small capsomere-interacting protein
R: Small capsomere-interacting protein
S: Major capsid protein
T: Major capsid protein
U: Major capsid protein
V: Major capsid protein
W: Major capsid protein
X: Major capsid protein
Y: Small capsomere-interacting protein
Z: Small capsomere-interacting protein
0: Small capsomere-interacting protein
1: Small capsomere-interacting protein
2: Small capsomere-interacting protein
3: Small capsomere-interacting protein
4: Major capsid protein
5: Triplex capsid protein 1
6: Triplex capsid protein 2
7: Triplex capsid protein 2
8: Triplex capsid protein 1
9: Triplex capsid protein 2
a: Triplex capsid protein 2
b: Triplex capsid protein 1
c: Triplex capsid protein 2
d: Triplex capsid protein 2
e: Triplex capsid protein 1
f: Triplex capsid protein 2
g: Triplex capsid protein 2
h: Triplex capsid protein 1
i: Triplex capsid protein 2
j: Triplex capsid protein 2
x 5


  • icosahedral pentamer
  • 16.3 MDa, 230 polymers
Theoretical massNumber of molelcules
Total (without water)16,304,066230
Polymers16,304,066230
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Major capsid protein
B: Major capsid protein
C: Major capsid protein
D: Major capsid protein
E: Major capsid protein
F: Major capsid protein
G: Small capsomere-interacting protein
H: Small capsomere-interacting protein
I: Small capsomere-interacting protein
J: Small capsomere-interacting protein
K: Small capsomere-interacting protein
L: Small capsomere-interacting protein
M: Major capsid protein
N: Major capsid protein
O: Major capsid protein
P: Small capsomere-interacting protein
Q: Small capsomere-interacting protein
R: Small capsomere-interacting protein
S: Major capsid protein
T: Major capsid protein
U: Major capsid protein
V: Major capsid protein
W: Major capsid protein
X: Major capsid protein
Y: Small capsomere-interacting protein
Z: Small capsomere-interacting protein
0: Small capsomere-interacting protein
1: Small capsomere-interacting protein
2: Small capsomere-interacting protein
3: Small capsomere-interacting protein
4: Major capsid protein
5: Triplex capsid protein 1
6: Triplex capsid protein 2
7: Triplex capsid protein 2
8: Triplex capsid protein 1
9: Triplex capsid protein 2
a: Triplex capsid protein 2
b: Triplex capsid protein 1
c: Triplex capsid protein 2
d: Triplex capsid protein 2
e: Triplex capsid protein 1
f: Triplex capsid protein 2
g: Triplex capsid protein 2
h: Triplex capsid protein 1
i: Triplex capsid protein 2
j: Triplex capsid protein 2
x 6


  • icosahedral 23 hexamer
  • 19.6 MDa, 276 polymers
Theoretical massNumber of molelcules
Total (without water)19,564,880276
Polymers19,564,880276
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

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Components

#1: Protein
Major capsid protein / MCP


Mass: 153574.188 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Details: Coded by gene ORF25. / Source: (natural) Human herpesvirus 8 / Variant: clone BAC16 / References: UniProt: D0UZN7
#2: Protein
Small capsomere-interacting protein


Mass: 18597.824 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Details: Coded by gene ORF65. / Source: (natural) Human herpesvirus 8 / Variant: clone BAC16 / References: UniProt: Q76RF4
#3: Protein
Triplex capsid protein 1


Mass: 36374.840 Da / Num. of mol.: 5 / Source method: isolated from a natural source
Details: Coded by gene ORF62. It is the monomer in the heterotrimeric triplex structure.
Source: (natural) Human herpesvirus 8 / Variant: clone BAC16 / References: UniProt: Q76RF6
#4: Protein
Triplex capsid protein 2


Mass: 34278.473 Da / Num. of mol.: 10 / Source method: isolated from a natural source
Details: Coded by gene ORF26. Forms the dimer in the heterotrimeric triplex structure.
Source: (natural) Human herpesvirus 8 / Variant: clone BAC16 / References: UniProt: C7E5A9
Has protein modificationY

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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: Human herpesvirus 8 / Type: VIRUS
Details: KSHV lytic replication was induced in an iSLK-puro cell line harboring the KSHV-BAC16 plasmid.
Entity ID: all / Source: NATURAL
Molecular weightValue: 200 MDa / Experimental value: NO
Source (natural)Organism: Human herpesvirus 8 / Strain: JSC-1
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Homo sapiens
Virus shellName: capsid / Diameter: 1300 nm / Triangulation number (T number): 16
Buffer solutionpH: 7.4
Buffer componentName: phosphate buffered saline / Formula: PBS
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Chamber temperature: 298 K
Details: The sample was manually blotted and frozen with a homemade plunger.

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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 FIELD / Nominal magnification: 14000 X / Calibrated magnification: 24271 X / Nominal defocus max: 2000 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 79 K
Image recordingAverage exposure time: 13 sec. / Electron dose: 25 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 8007
Image scansSampling size: 2.5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 26 / Used frames/image: 1-26

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Processing

SoftwareName: PHENIX / Version: dev_2875: / Classification: refinement
EM software
IDNameVersionCategory
1ETHANparticle selection
2Leginonimage acquisition
4CTFFIND3CTF correction
9IMIRSinitial Euler assignment
10IMIRSfinal Euler assignment
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 44343
Details: Particles were boxed with ETHAN, and then manually examined.
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25315 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008219357
ELECTRON MICROSCOPYf_angle_d1.119298106
ELECTRON MICROSCOPYf_dihedral_angle_d6.325132195
ELECTRON MICROSCOPYf_chiral_restr0.05933590
ELECTRON MICROSCOPYf_plane_restr0.00838789

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