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- EMDB-3281: Antigenic and cryo-electron microscopy structure analysis of a ch... -

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Entry
Database: EMDB / ID: EMD-3281
TitleAntigenic and cryo-electron microscopy structure analysis of a chimeric sapovirus capsid
Map dataChimeric human sapovirus capsid
Sample
  • Sample: Chimera human sapovirus capsid
  • Virus: Sapovirus
Keywordsnon-envelope virus / VLP / calicivirus
Biological speciesSapovirus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.5 Å
AuthorsMiyazaki N / Taylor DW / Hansman GS / Murata K
CitationJournal: J Virol / Year: 2015
Title: Antigenic and Cryo-Electron Microscopy Structure Analysis of a Chimeric Sapovirus Capsid.
Authors: Naoyuki Miyazaki / David W Taylor / Grant S Hansman / Kazuyoshi Murata /
Abstract: The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S ...The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S domain forms a scaffold surrounding the nucleic acid, while the P domains form a homodimer that interacts with receptors. The P domain is further subdivided into two subdomains, termed P1 and P2. The P2 subdomain is likely an insertion in the P1 subdomain; consequently, the P domain is divided into the P1-1, P2, and P1-2 subdomains. In order to investigate capsid antigenicity, N-terminal (N-term)/S/P1-1 and P2/P1-2 were switched between two sapovirus genotypes GI.1 and GI.5. The chimeric VP1 constructs were expressed in insect cells and were shown to self-assemble into virus-like particles (VLPs) morphologically similar to the parental VLPs. Interestingly, the chimeric VLPs had higher levels of cross-reactivities to heterogeneous antisera than the parental VLPs. In order to better understand the antigenicity from a structural perspective, we determined an intermediate-resolution (8.5-Å) cryo-electron microscopy (cryo-EM) structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that the P domain dimers were raised slightly (∼5 Å) above the S domain. The VP1 homology model allowed us predict the S domain (67-229) and P1-1 (229-280), P2 (281-447), and P1-2 (448-567) subdomains. Our results suggested that the raised P dimers might expose immunoreactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes.
IMPORTANCE: We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against ...IMPORTANCE: We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera than the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5-Å resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. The overall structure of Yokote/Mc114 contained features common among other caliciviruses. We showed that the P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had fewer interactions.
History
DepositionDec 14, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseFeb 10, 2016-
UpdateFeb 8, 2017-
Current statusFeb 8, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2681
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2681
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3281.png

Downloads & links

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Map

FileDownload / File: emd_3281.map.gz / Format: CCP4 / Size: 127.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationChimeric human sapovirus capsid
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.62 Å/pix.
x 325 pix.
= 526.5 Å		1.62 Å/pix.
x 325 pix.
= 526.5 Å		1.62 Å/pix.
x 325 pix.
= 526.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.62 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2681.0 / Movie #1: 2681
Minimum - Maximum-5821.080566409999847 - 12061.328125
Average (Standard dev.)384.011596680000025 (±1160.938354489999938)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-162-162-162
Dimensions325325325
Spacing325325325
CellA=B=C: 526.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.621.621.62
M x/y/z325325325
origin x/y/z0.0000.0000.000
length x/y/z526.500526.500526.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-162-162-162
NC/NR/NS325325325
D min/max/mean-5821.08112061.328384.012

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Supplemental data

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Sample components

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Entire : Chimera human sapovirus capsid

EntireName: Chimera human sapovirus capsid
Components
  • Sample: Chimera human sapovirus capsid
  • Virus: Sapovirus

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Supramolecule #1000: Chimera human sapovirus capsid

SupramoleculeName: Chimera human sapovirus capsid / type: sample / ID: 1000 / Oligomeric state: icosahedral / Number unique components: 1
Molecular weightExperimental: 10 MDa / Theoretical: 10 MDa

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Supramolecule #1: Sapovirus

SupramoleculeName: Sapovirus / type: virus / ID: 1 / Name.synonym: human sapovirus / NCBI-ID: 95341 / Sci species name: Sapovirus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes / Syn species name: human sapovirus
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: unidentified baculovirus
Molecular weightExperimental: 10 MDa / Theoretical: 10 MDa
Virus shellShell ID: 1 / Diameter: 38 Å / T number (triangulation number): 3

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

StainingType: NEGATIVE / Details: ice-embedding
GridDetails: quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeJEOL 2200FS
TemperatureMin: 90 K / Max: 105 K / Average: 100 K
Specialist opticsEnergy filter - Name: Omega-type / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 15.0 eV
DateJul 15, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 80 / Average electron dose: 20 e/Å2 / Od range: 5 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 91463 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 6000

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