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- EMDB-36483: CryoEM structure of isNS1 in complex with Fab56.2 -

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Basic information

Entry
Database: EMDB / ID: EMD-36483
TitleCryoEM structure of isNS1 in complex with Fab56.2
Map data
Sample
  • Complex: Ternary complex of Fab56.2 with dengue virus nonstructural protein 1 and High Density Lipoprotein
    • Complex: isNS1ts
      • Other: core protein
    • Complex: Fab56.2
      • Other: Fab56.2 Heavy Chain
      • Other: Fab56.2 Light Chain
KeywordsFlavivirus / Dengue / NS1 / HDL / VIRAL PROTEIN
Biological speciesDengue virus type 2 / Mus musculus (house mouse) / dengue virus type 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsChew BLA / Luo D
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)T2EP30220-0020 Singapore
Citation
Journal: Elife / Year: 2024
Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein.
Authors: Bing Liang Alvin Chew / A N Qi Ngoh / Wint Wint Phoo / Kitti Wing Ki Chan / Zheng Ser / Nikhil K Tulsian / Shiao See Lim / Mei Jie Grace Weng / Satoru Watanabe / Milly M Choy / Jenny Low / ...Authors: Bing Liang Alvin Chew / A N Qi Ngoh / Wint Wint Phoo / Kitti Wing Ki Chan / Zheng Ser / Nikhil K Tulsian / Shiao See Lim / Mei Jie Grace Weng / Satoru Watanabe / Milly M Choy / Jenny Low / Eng Eong Ooi / Christiane Ruedl / Radoslaw M Sobota / Subhash G Vasudevan / Dahai Luo /
Abstract: Severe dengue infections are characterized by endothelial dysfunction shown to be associated with the secreted nonstructural protein 1 (sNS1), making it an attractive vaccine antigen and ...Severe dengue infections are characterized by endothelial dysfunction shown to be associated with the secreted nonstructural protein 1 (sNS1), making it an attractive vaccine antigen and biotherapeutic target. To uncover the biologically relevant structure of sNS1, we obtained infection-derived sNS1 (isNS1) from dengue virus (DENV)-infected Vero cells through immunoaffinity purification instead of recombinant sNS1 (rsNS1) overexpressed in insect or mammalian cell lines. We found that isNS1 appeared as an approximately 250 kDa complex of NS1 and ApoA1 and further determined the cryoEM structures of isNS1 and its complex with a monoclonal antibody/Fab. Indeed, we found that the major species of isNS1 is a complex of the NS1 dimer partially embedded in a high-density lipoprotein (HDL) particle. Crosslinking mass spectrometry studies confirmed that the isNS1 interacts with the major HDL component ApoA1 through interactions that map to the NS1 wing and hydrophobic domains. Furthermore, our studies demonstrated that the sNS1 in sera from DENV-infected mice and a human patient form a similar complex as isNS1. Our results report the molecular architecture of a biological form of sNS1, which may have implications for the molecular pathogenesis of dengue.
#2: Journal: Elife / Year: 2023
Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein.
Authors: Chew BLA / Ngoh AQ / Phoo WW / Chan KWK / Ser Z / Tulsian NK / Lim SS / Weng MJG / Watanabe S / Choy MM / Low JG / Ooi EE / Ruedl C / Sobota RM / Vasudevan SG / Luo D
History
DepositionJun 12, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36483.png

Downloads & links

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Map

FileDownload / File: emd_36483.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 416 pix.
= 356.928 Å		0.86 Å/pix.
x 416 pix.
= 356.928 Å		0.86 Å/pix.
x 416 pix.
= 356.928 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.858 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.9048512 - 1.8042191
Average (Standard dev.)-0.0013161432 (±0.027252153)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 356.92798 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_36483_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_36483_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of Fab56.2 with dengue virus nonstructural protei...

EntireName: Ternary complex of Fab56.2 with dengue virus nonstructural protein 1 and High Density Lipoprotein
Components
  • Complex: Ternary complex of Fab56.2 with dengue virus nonstructural protein 1 and High Density Lipoprotein
    • Complex: isNS1ts
      • Other: core protein
    • Complex: Fab56.2
      • Other: Fab56.2 Heavy Chain
      • Other: Fab56.2 Light Chain

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Supramolecule #1: Ternary complex of Fab56.2 with dengue virus nonstructural protei...

SupramoleculeName: Ternary complex of Fab56.2 with dengue virus nonstructural protein 1 and High Density Lipoprotein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: sNS1 derived from the supernatant of vero cells infected by DENV2 EDEN strain. Fab56.2 fragment was added in exogeneously, generated through proteolytic cleavage of antibody derived from hybridoma supernatant.

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Supramolecule #2: isNS1ts

SupramoleculeName: isNS1ts / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Dengue virus 2 (EDEN2 strain, T164S mutant) secreted NS1 from infected vero cells
Source (natural)Organism: Dengue virus type 2 / Strain: EDEN2

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Supramolecule #3: Fab56.2

SupramoleculeName: Fab56.2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: ab56.2 fragment was generated through proteolytic cleavage of antibody derived from hybridoma supernatant.
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: core protein

MacromoleculeName: core protein / type: other / ID: 1
Details: sNS1 derived from the supernatant of vero cells infected by DENV2 EDEN strain. Fab56.2 fragment was added in exogeneously, generated through proteolytic cleavage of antibody derived from hybridoma supernatant.
Classification: other
Source (natural)Organism: dengue virus type 2 / Strain: EDEN2
SequenceString: DSGCVVSWKN KELKCGSGIF ITDNVHTWTE QYKFQPESPS KLASAIQKAH EEGICGIRSV TRLENLMWKQ ITPELNHILS ENEVKMTIMT GDIKGIMQAG KRSLRPQPTE LKYSWKAWGK AKMLSTELHN HTFLIDGPET AECPNTNRAW NSLEVEDYGF GVFTTNIWLK ...String:
DSGCVVSWKN KELKCGSGIF ITDNVHTWTE QYKFQPESPS KLASAIQKAH EEGICGIRSV TRLENLMWKQ ITPELNHILS ENEVKMTIMT GDIKGIMQAG KRSLRPQPTE LKYSWKAWGK AKMLSTELHN HTFLIDGPET AECPNTNRAW NSLEVEDYGF GVFTTNIWLK LKERQDVSCD SKLMSAAIKD NRAVHADMGY WIESALNDTW KIEKASFIEV KSCHWPKSHT LWSNGVLESE MIIPKNFAGP VSQHNYRPGY HTQTAGPWHL GRLEMDFDFC EGTTVVVTED CGNRGPSLRT TTASGKLITE WCCRSCTLPP LRYRGEDGCW YGMEIRPLKE KEENLVNSLV TA

GENBANK: GENBANK: EU081177

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Macromolecule #2: Fab56.2 Heavy Chain

MacromoleculeName: Fab56.2 Heavy Chain / type: other / ID: 2 / Details: mouse hybridoma / Classification: other
Source (natural)Organism: Mus musculus (house mouse)
SequenceString: ALEVQLQESG PGLVKPSQSL SFTWSVIGYS ITSGYYWKWI RQFSGNKLEW MGYIRYDGSN NYNPSLKNRI SIIRDTFKNQ FFLKLNFVTA GDTATYYCAN LYYYSSSPGW FPYWGQGTLV TVFAASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL ...String:
ALEVQLQESG PGLVKPSQSL SFTWSVIGYS ITSGYYWKWI RQFSGNKLEW MGYIRYDGSN NYNPSLKNRI SIIRDTFKNQ FFLKLNFVTA GDTATYYCAN LYYYSSSPGW FPYWGQGTLV TVFAASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GPQTYICNVN HKPSNTKVDK KVEPKSCD

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Macromolecule #3: Fab56.2 Light Chain

MacromoleculeName: Fab56.2 Light Chain / type: other / ID: 3 / Details: mouse hybridoma / Classification: other
Source (natural)Organism: Mus musculus (house mouse)
SequenceString: AELDIVMTQS PAIMSASLGE EITLTCSASS SVSYMHWYQQ KSGTSPKLLI YSTSNLASGV PSRFSGSGSG TFYSLTISSV EAEDAADYYC HQWSSWTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD ...String:
AELDIVMTQS PAIMSASLGE EITLTCSASS SVSYMHWYQQ KSGTSPKLLI YSTSNLASGV PSRFSGSGSG TFYSLTISSV EAEDAADYYC HQWSSWTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSLPVTKSFN RGEC

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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

Concentration0.35 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: NS1 dimer model generated using AF2
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 164009
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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