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- EMDB-6908: CryoEM structure of HPV31 L1-only VLP -

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Basic information

Entry
Database: EMDB / ID: EMD-6908
TitleCryoEM structure of HPV31 L1-only VLP
Map data
Sample
  • Virus: Human papillomavirus type 31
Biological speciesHuman papillomavirus type 31
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsLi SW / Li ZH
CitationJournal: Protein Expr Purif / Year: 2017
Title: Stop codon mutagenesis for homogenous expression of human papillomavirus L1 protein in Escherichia coli.
Authors: Daning Wang / Fei Fan / Zhihai Li / Xinlin Liu / Shuo Song / Shuangping Wei / Maozhou He / Yahua Lin / Zhongyi Li / Minxi Wei / Hai Yu / Ying Gu / Shaowei Li / Ningshao Xia /
Abstract: Human papillomavirus (HPV) is widely accepted to be the major causative pathogen of cervical cancer, warts, and other epithelial tumors. Virus infection and subsequent disease development can be ...Human papillomavirus (HPV) is widely accepted to be the major causative pathogen of cervical cancer, warts, and other epithelial tumors. Virus infection and subsequent disease development can be prevented by vaccination with HPV vaccines derived from eukaryotic expression systems. Here, we report the soluble expression of the major capsid protein L1 of HPV31, a dominant carcinogenic HPV genotype, in Escherichia coli. HPV31 L1 protein and its elongated form (L1+) were observed in SDS-PAGE and CE-SDS analysis, generated by the native HPV31 L1 gene with a TAA stop codon. Replacing the TAA with TAG but not TGA could completely terminate protein translation. Mass spectrometry sequencing showed that L1+ comprised L1 with a C-terminal extension of 38 amino acids (aa). RNA folding analysis revealed that the unfaithful L1+ expression may result from translational read-through, as TAG is more stable and accessible than the other stop codons. The 38-aa elongated fragment perturbs self-assembly of HPV31 L1+, as shown in size and morphology analyses. By 3D cryo-electron microscopy structure determination, we show self-assembly of purified HPV31 L1 (TAG) VLPs into T = 7 icosahedral symmetry particles, resembling the native HPV virion. Finally, through additional characterization and antigenicity/immunogenicity assays, we verified that the E.coli-derived HPV31 VLPs are an ideal immunogen for HPV vaccine development. Our findings outline a codon optimization stratagem for protein expression and provide a method for the in-depth investigation of prokaryotic translation regulation.
History
DepositionFeb 10, 2018-
Header (metadata) releaseFeb 27, 2019-
Map releaseFeb 27, 2019-
UpdateFeb 27, 2019-
Current statusFeb 27, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6908.png

Downloads & links

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Map

FileDownload / File: emd_6908.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 4.44 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-6.453121 - 7.375142
Average (Standard dev.)0.000000001210974 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-92-92-92
Dimensions180180180
Spacing180180180
CellA=B=C: 799.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.444.444.44
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z799.200799.200799.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ364364364
MAP C/R/S123
start NC/NR/NS-92-92-92
NC/NR/NS180180180
D min/max/mean-6.4537.3750.000

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Supplemental data

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Sample components

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Entire : Human papillomavirus type 31

EntireName: Human papillomavirus type 31
Components
  • Virus: Human papillomavirus type 31

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Supramolecule #1: Human papillomavirus type 31

SupramoleculeName: Human papillomavirus type 31 / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 10585 / Sci species name: Human papillomavirus type 31 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host systemOrganism: Escherichia coli (E. coli)

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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: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON I (4k x 4k) / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: COMMON LINE
Final angle assignmentType: COMMON LINE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 536

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