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- EMDB-7047: CryoEM structure and atomic model of the Kaposi's sarcoma-associa... -

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Basic information

Entry
Database: EMDB / ID: EMD-7047
TitleCryoEM structure and atomic model of the Kaposi's sarcoma-associated herpesvirus capsid
Map dataCryoEM structure and atomic model of the Kaposi's sarcoma-associated herpesvirus capsid
Sample
  • Virus: Human herpesvirus 8
    • Protein or peptide: Major capsid protein
    • Protein or peptide: Small capsomere-interacting protein
    • Protein or peptide: Triplex capsid protein 1
    • Protein or peptide: Triplex capsid protein 2
Function / homology
Function and homology information


T=16 icosahedral viral capsid / viral capsid assembly / viral process / viral capsid / host cell nucleus / structural molecule activity / DNA binding
Similarity search - Function
Gammaherpesvirus capsid / Gammaherpesvirus capsid protein / Herpesvirus capsid protein 2 / Herpesvirus capsid shell protein 1 / Herpesvirus VP23 like capsid protein / Herpesvirus capsid shell protein VP19C / 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 speciesHHV-8 (virus) / Human herpesvirus 8
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsDai XH / Gong DY / Sun R / Zhou ZH
Funding support United States, 8 items
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 Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA177322 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA091791 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1U24GM116792 United States
National Institutes of Health/Office of the Director1S10OD018111 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, 2017-
Header (metadata) releaseNov 8, 2017-
Map releaseNov 8, 2017-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6b43
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6b43
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7047.png

Downloads & links

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Map

FileDownload / File: emd_7047.map.gz / Format: CCP4 / Size: 7.8 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure and atomic model of the Kaposi's sarcoma-associated herpesvirus capsid
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-11.5964775 - 15.720491
Average (Standard dev.)0.008491504 (±1.0703465)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-640-640-640
Dimensions128012801280
Spacing128012801280
CellA=B=C: 1318.3999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z128012801280
origin x/y/z0.0000.0000.000
length x/y/z1318.4001318.4001318.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-640-640-640
NC/NR/NS128012801280
D min/max/mean-11.59615.7200.008

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Supplemental data

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Sample components

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Entire : Human herpesvirus 8

EntireName: Human herpesvirus 8
Components
  • Virus: Human herpesvirus 8
    • Protein or peptide: Major capsid protein
    • Protein or peptide: Small capsomere-interacting protein
    • Protein or peptide: Triplex capsid protein 1
    • Protein or peptide: Triplex capsid protein 2

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Supramolecule #1: Human herpesvirus 8

SupramoleculeName: Human herpesvirus 8 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: KSHV lytic replication was induced in an iSLK-puro cell line harboring the KSHV-BAC16 plasmid.
NCBI-ID: 37296 / Sci species name: Human herpesvirus 8 / Sci species strain: JSC-1 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 200 MDa
Virus shellShell ID: 1 / Name: capsid / Diameter: 1300.0 Å / T number (triangulation number): 16

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Macromolecule #1: Major capsid protein

MacromoleculeName: Major capsid protein / type: protein_or_peptide / ID: 1 / Details: Coded by gene ORF25. / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: HHV-8 (virus)
Molecular weightTheoretical: 153.574188 KDa
SequenceString: MEATLEQRPF PYLATEANLL TQIKESAADG LFKSFQLLLG KDAREGSVRF EALLGVYTNV VEFVKFLETA LAAACVNTEF KDLRRMIDG KIQFKISMPT IAHGDGRRPN KQRQYIVMKA CNKHHIGAEI ELAAADIELL FAEKETPLDF TEYAGAIKTI T SALQFGMD ...String:
MEATLEQRPF PYLATEANLL TQIKESAADG LFKSFQLLLG KDAREGSVRF EALLGVYTNV VEFVKFLETA LAAACVNTEF KDLRRMIDG KIQFKISMPT IAHGDGRRPN KQRQYIVMKA CNKHHIGAEI ELAAADIELL FAEKETPLDF TEYAGAIKTI T SALQFGMD ALERGLVDTV LAVKLRHAPP VFILKTLGDP VYSERGLKKA VKSDMVSMFK AHLIEHSFFL DKAELMTRGK QY VLTMLSD MLAAVCEDTV FKGVSTYTTA SGQQVAGVLE TTDSVMRRLM NLLGQVESAM SGPAAYASYV VRGANLVTAV SYG RAMRNF EQFMARIVDH PNALPSVEGD KAALADGHDE IQRTRIAASL VKIGDKFVAI ESLQRMYNET QFPCPLNRRI QYTY FFPVG LHLPVPRYST SVSVRGVESP AIQSTETWVV NKNNVPLCFG YQNALKSICH PRMHNPTQSA QALNQAFPDP DGGHG YGLR YEQTPNMNLF RTFHQYYMGK NVAFVPDVAQ KALVTTEDLL HPTSHRLLRL EVHPFFDFFV HPCPGARGSY RATHRT MVG NIPQPLAPRE FQESRGAQFD AVTNMTHVID QLTIDVIQET AFDPAYPLFC YVIEAMIHGQ EEKFVMNMPL IALVIQT YW VNSGKLAFVN SYHMVRFICT HMGNGSIPKE AHGHYRKILG ELIALEQALL KLAGHETVGR TPITHLVSAL LDPHLLPP F AYHDVFTDLM QKSSRQPIIK IGDQNYDNPQ NRATFINLRG RMEDLVNNLV NIYQTRVNED HDERHVLDVA PLDENDYNP VLEKLFYYVL MPVCSNGHMC GMGVDYQNVA LTLTYNGPVF ADVVNAQDDI LLHLENGTLK DILQAGDIRP TVDMIRVLCT SFLTCPFVT QAARVITKRD PAQSFATHEY GKDVAQTVLV NGFGAFAVAD RSREAAETMF YPVPFNKLYA DPLVAATLHP L LANYVTRL PNQRNAVVFN VPSNLMAEYE EWHKSPVAAY AASCQATPGA ISAMVSMHQK LSAPSFICQA KHRMHPGFAM TV VRTDEVL AEHILYCSRA STSMFVGLPS VVRREVRSDA VTFEITHEIA SLHTALGYSS VIAPAHVAAI TTDMGVHCQD LFM IFPGDA YQDRQLHDYI KMKAGVQTGP PGNRMDHVGY AAGVPRCENL PGLSHGQLAT CEIIPTPVTS DVAYFQTPSN PRGR AACVV SCDAYSNESA ERLLYDHSIP DPAYECRSTN NPWASQRGSL GDVLYNITFR QTALPGMYSP CRQFFHKEDI MRYNR GLYT LVNEYSARLA GAPATSTTDL QYVVVNGTDV FLDQPCHMLQ EAYPTLAASH RVMLDEYMSN KQTHAPVHMG QYLIEE VAP MKRLLKLGNK VVY

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Macromolecule #2: Small capsomere-interacting protein

MacromoleculeName: Small capsomere-interacting protein / type: protein_or_peptide / ID: 2 / Details: Coded by gene ORF65. / Number of copies: 15 / Enantiomer: LEVO
Source (natural)Organism: HHV-8 (virus)
Molecular weightTheoretical: 18.597824 KDa
SequenceString:
MSNFKVRDPV IQERLDHDYA HHPLVARMNT LDQGNMSQAE YLVQKRHYLV FLIAHHYYEA YLRRMGGIQR RDHLQTLRDQ KPRERADRV SAASAYDAGT FTVPSRPGPA SGTTPGGQDS LGVSGSSITT LSSGPHSLSP ASDILTTLSS TTETAAPAVA D ARKPPSGK KK

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Macromolecule #3: Triplex capsid protein 1

MacromoleculeName: Triplex capsid protein 1 / type: protein_or_peptide / ID: 3
Details: Coded by gene ORF62. It is the monomer in the heterotrimeric triplex structure.
Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: HHV-8 (virus)
Molecular weightTheoretical: 36.37484 KDa
SequenceString: MKVQAENAAR LGRQVLGLLP PPTHRVSLTR GPEFARGVRD LLSKYAASTR PTVGSLHEAL RQAPFRQPTY GDFLVYSQTF SPQEPLGTF LFSFKQEDNG SSMDMLLTPT SLFMLSGMEA AKAPQTHKVA GVWYGSGSGL ADFIPNLSEL MDTGEFHTLL T PVGPMVQS ...String:
MKVQAENAAR LGRQVLGLLP PPTHRVSLTR GPEFARGVRD LLSKYAASTR PTVGSLHEAL RQAPFRQPTY GDFLVYSQTF SPQEPLGTF LFSFKQEDNG SSMDMLLTPT SLFMLSGMEA AKAPQTHKVA GVWYGSGSGL ADFIPNLSEL MDTGEFHTLL T PVGPMVQS VHSTFVTKVT SAMKGVGLAR DEPRAHVGLT LPCDMLVDLD ESCPMVQRRE PAGLNVTIYA SLVYLRVNQR PS MALTFFQ SGKGFAEVVA MIKDHFTDVI RTKYIQLRHE LYINRLVFGA VCTLGTVPFD SHPVHQSLNV KGTSLPVLVF ANF EAACGP WTVFL

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Macromolecule #4: Triplex capsid protein 2

MacromoleculeName: Triplex capsid protein 2 / type: protein_or_peptide / ID: 4
Details: Coded by gene ORF26. Forms the dimer in the heterotrimeric triplex structure.
Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: HHV-8 (virus)
Molecular weightTheoretical: 34.278473 KDa
SequenceString: MALDKSIVVN LTSRLFADEL AALQSKIGSV LPLGDCHRLQ NIQALGLGCV CSRETSPDYI QIMQYLSKCT LAVLEEVRPD SLRLTRMDP SDNLQIKNVY APFFQWDSNT QLAVLPPLFS RKDSTIVLES NGFDIVFPMV VPQQLGHAIL QQLLVYHIYS K ISAGAPGD ...String:
MALDKSIVVN LTSRLFADEL AALQSKIGSV LPLGDCHRLQ NIQALGLGCV CSRETSPDYI QIMQYLSKCT LAVLEEVRPD SLRLTRMDP SDNLQIKNVY APFFQWDSNT QLAVLPPLFS RKDSTIVLES NGFDIVFPMV VPQQLGHAIL QQLLVYHIYS K ISAGAPGD VNMAELDLYT TNVSFMGRTY RLDVDNTDPR TALRVLDDLS MYLCILSALV PRGCLRLLTA LVRHDRHPLT EV FEGVVPD EVTRIDLDQL SVPDDITRMR VMFSYLQSLS SIFNLGPRLH VYAYSAETLA ASCWYSPR

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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: 7.4 / Component - Formula: PBS / Component - Name: phosphate buffered saline
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER
Details: The sample was manually blotted and frozen with a homemade plunger..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 24271 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 14000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 79.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Sampling interval: 2.5 µm / Digitization - Frames/image: 1-26 / Number grids imaged: 3 / Number real images: 8007 / Average exposure time: 13.0 sec. / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 44343
Details: Particles were boxed with ETHAN, and then manually examined.
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: EMDB MAP
EMDB ID:

6038

Initial angle assignmentType: COMMON LINE / Software - Name: IMIRS
Final angle assignmentType: COMMON LINE / Software - Name: IMIRS
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 25315

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