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- EMDB-26740: Ligand-free Lassa GPC Trimer with C1 Symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-26740
TitleLigand-free Lassa GPC Trimer with C1 Symmetry
Map dataSharpened map
Sample
  • Complex: Lassa GPC Trimer
    • Protein or peptide: Lassa GPC Trimer
KeywordsGPC / GP1 / GP2 / Lassa / Arenavirus / Cleavage intermediate / VIRAL PROTEIN
Biological speciesLassa virus Josiah
Methodsingle particle reconstruction / cryo EM / Resolution: 6.58 Å
AuthorsGorman J / Kwong PD
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
Simons FoundationSF349247 United States
CitationJournal: Nat Commun / Year: 2024
Title: Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.
Authors: Jason Gorman / Crystal Sao-Fong Cheung / Zhijian Duan / Li Ou / Maple Wang / Xuejun Chen / Cheng Cheng / Andrea Biju / Yaping Sun / Pengfei Wang / Yongping Yang / Baoshan Zhang / Jeffrey C ...Authors: Jason Gorman / Crystal Sao-Fong Cheung / Zhijian Duan / Li Ou / Maple Wang / Xuejun Chen / Cheng Cheng / Andrea Biju / Yaping Sun / Pengfei Wang / Yongping Yang / Baoshan Zhang / Jeffrey C Boyington / Tatsiana Bylund / Sam Charaf / Steven J Chen / Haijuan Du / Amy R Henry / Tracy Liu / Edward K Sarfo / Chaim A Schramm / Chen-Hsiang Shen / Tyler Stephens / I-Ting Teng / John-Paul Todd / Yaroslav Tsybovsky / Raffaello Verardi / Danyi Wang / Shuishu Wang / Zhantong Wang / Cheng-Yan Zheng / Tongqing Zhou / Daniel C Douek / John R Mascola / David D Ho / Mitchell Ho / Peter D Kwong /
Abstract: Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. ...Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.
History
DepositionApr 25, 2022-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26740.png

Downloads & links

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Map

FileDownload / File: emd_26740.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 1.07325 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.35793543 - 1.2147697
Average (Standard dev.)0.0022093987 (±0.057279762)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 274.752 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26740_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_26740_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_26740_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_26740_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Lassa GPC Trimer

EntireName: Lassa GPC Trimer
Components
  • Complex: Lassa GPC Trimer
    • Protein or peptide: Lassa GPC Trimer

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Supramolecule #1: Lassa GPC Trimer

SupramoleculeName: Lassa GPC Trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lassa virus Josiah

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Macromolecule #1: Lassa GPC Trimer

MacromoleculeName: Lassa GPC Trimer / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Lassa virus Josiah
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TSLYKGVYEL QTLELNMETL NMTMPLSCTK NNSHHYIMVG NETGLELTLT NTSIINHKFC NLSDAHKKNL YDHALMSIIS TFHLSIPNFN QYEAMSCDFN GGKISVQYNL SHSYAGDAAN HCGTVANGVL QTFMRMAWGG SYIALDSGGC GNWDCIMTSY QYLIIQNTTW ...String:
TSLYKGVYEL QTLELNMETL NMTMPLSCTK NNSHHYIMVG NETGLELTLT NTSIINHKFC NLSDAHKKNL YDHALMSIIS TFHLSIPNFN QYEAMSCDFN GGKISVQYNL SHSYAGDAAN HCGTVANGVL QTFMRMAWGG SYIALDSGGC GNWDCIMTSY QYLIIQNTTW EDHCQFSRPS PIGYLGLLSQ RTRDIYISRR RRGTFTWTLS DSEGKDTPGG YCLTRWMLIE AELKCFGNTA VAKCNEKHDE EFCDMLRLFD FNKQAIQRCK APAQMSIQLI NKAVNALIND QLIMKNHLRD IMGIPYCNYS KYWYLNHTTT GRTSLPKCWL VSNGSYLNET HFSDDIEQQA DNMITEMLQK EGGGYIPEAP RDGQAYVRKD GEWVLLSTFL GGLVPR

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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

Concentration3 mg/mL
BufferpH: 7.4 / Details: PBS
GridModel: C-flat-1.2/1.3 / Material: COPPER / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Average electron dose: 56.52 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.58 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 43577

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