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

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Basic information

Entry
Database: EMDB / ID: 6908
TitleCryoEM structure of HPV31 L1-only VLP
Map data
SampleHuman papillomavirus type 31:
virus
SourceHuman papillomavirus type 31
Methodsingle particle reconstruction / cryo EM / 20 Å resolution
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.
DateDeposition: Feb 10, 2018 / Header (metadata) release: Feb 27, 2019 / Map release: Feb 27, 2019 / Last update: Feb 27, 2019

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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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Structure viewerEM map:
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Supplemental images

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Map

Fileemd_6908.map.gz (map file in CCP4 format, 23329 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
180 pix
4.44 Å/pix.
= 799.2 Å
180 pix
4.44 Å/pix.
= 799.2 Å
180 pix
4.44 Å/pix.
= 799.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.44 Å
Density
Contour Level:1.0 (by author), 1 (movie #1):
Minimum - Maximum-6.453121 - 7.375142
Average (Standard dev.)0.000000001210974 (1.0)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions180180180
Origin-92.0-92.0-92.0
Limit87.087.087.0
Spacing180180180
CellA=B=C: 799.2 Å
α=β=γ: 90.0 deg.

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 / Number of components: 1

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Component #1: virus, Human papillomavirus type 31

VirusName: Human papillomavirus type 31 / Class: VIRUS-LIKE PARTICLE / Empty: Yes / Enveloped: No / Isolate: OTHER
SpeciesSpecies: Human papillomavirus type 31
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F30
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: SPOT SCAN
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON I (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 536
3D reconstructionResolution: 2 Å / Resolution method: FSC 0.5 CUT-OFF

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