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- EMDB-50635: Unmasked HIV-1 envelope trimer gp140 SpyTag SpyCatcher mi3 nanopa... -

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Basic information

Entry
Database: EMDB / ID: EMD-50635
TitleUnmasked HIV-1 envelope trimer gp140 SpyTag SpyCatcher mi3 nanoparticle.
Map dataOverall map without masking or classification.
Sample
  • Complex: CAP255 gp140 SpyTag-SpyCatcher mi3 nanoparticle
    • Protein or peptide: HIV-1 Envelope trimer gp140 SpyTag SpyCatcher mi3
KeywordsVaccine / HIV-1 / mi3 / Nanoparticle / VLP / IMMUNE SYSTEM
Biological speciesThermotoga maritima (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsWoodward JD / Malebo K / Chapman R
Funding support South Africa, United States, 2 items
OrganizationGrant numberCountry
South African Medical Research Council self-initiated research grant (SAMRC SIR Grant) South Africa
Chan Zuckerberg Initiative2021-240494 United States
CitationJournal: Vaccines (Basel) / Year: 2024
Title: Development of a Two-Component Nanoparticle Vaccine Displaying an HIV-1 Envelope Glycoprotein that Elicits Tier 2 Neutralising Antibodies.
Authors: Kegomoditswe Malebo / Jeremy Woodward / Phindile Ximba / Qiniso Mkhize / Sanele Cingo / Thandeka Moyo-Gwete / Penny L Moore / Anna-Lise Williamson / Rosamund Chapman /
Abstract: Despite treatment and other interventions, an effective prophylactic HIV vaccine is still an essential goal in the control of HIV. Inducing robust and long-lasting antibody responses is one of the ...Despite treatment and other interventions, an effective prophylactic HIV vaccine is still an essential goal in the control of HIV. Inducing robust and long-lasting antibody responses is one of the main targets of an HIV vaccine. The delivery of HIV envelope glycoproteins (Env) using nanoparticle (NP) platforms has been shown to elicit better immunogenicity than soluble HIV Env. In this paper, we describe the development of a nanoparticle-based vaccine decorated with HIV Env using the SpyCatcher/SpyTag system. The Env utilised in this study, CAP255, was derived from a transmitted founder virus isolated from a patient who developed broadly neutralising antibodies. Negative stain and cryo-electron microscopy analyses confirmed the assembly and stability of the mi3 into uniform icosahedral NPs surrounded by regularly spaced CAP255 gp140 Env trimers. A three-dimensional reconstruction of CAP255 gp140 SpyTag-SpyCatcher mi3 clearly showed Env trimers projecting from the centre of each of the pentagonal dodecahedral faces of the NP. To our knowledge, this is the first study to report the formation of SpyCatcher pentamers on the dodecahedral faces of mi3 NPs. To investigate the immunogenicity, rabbits were primed with two doses of DNA vaccines expressing the CAP255 gp150 and a mosaic subtype C Gag and boosted with three doses of the NP-developed autologous Tier 2 CAP255 neutralising antibodies (Nabs) and low levels of heterologous CAP256SU NAbs.
History
DepositionJun 12, 2024-
Header (metadata) releaseOct 9, 2024-
Map releaseOct 9, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50635.png

Downloads & links

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Map

FileDownload / File: emd_50635.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOverall map without masking or classification.
Projections & slices

Image control

Size
Brightness
Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
1.66 Å/pix.
x 288 pix.
= 478.656 Å		1.66 Å/pix.
x 288 pix.
= 478.656 Å		1.66 Å/pix.
x 288 pix.
= 478.656 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.662 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00386
Minimum - Maximum-0.0066859364 - 0.04443644
Average (Standard dev.)0.0010817726 (±0.003140693)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 478.65598 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_50635_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_50635_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CAP255 gp140 SpyTag-SpyCatcher mi3 nanoparticle

EntireName: CAP255 gp140 SpyTag-SpyCatcher mi3 nanoparticle
Components
  • Complex: CAP255 gp140 SpyTag-SpyCatcher mi3 nanoparticle
    • Protein or peptide: HIV-1 Envelope trimer gp140 SpyTag SpyCatcher mi3

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Supramolecule #1: CAP255 gp140 SpyTag-SpyCatcher mi3 nanoparticle

SupramoleculeName: CAP255 gp140 SpyTag-SpyCatcher mi3 nanoparticle / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: CAP255 gp140 SpyTag was expressed in HEK293T cells and purified. SpyCatcher mi3 was expressed in
Source (natural)Organism: Thermotoga maritima (bacteria)
Molecular weightTheoretical: 4.75 MDa

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Macromolecule #1: HIV-1 Envelope trimer gp140 SpyTag SpyCatcher mi3

MacromoleculeName: HIV-1 Envelope trimer gp140 SpyTag SpyCatcher mi3 / type: protein_or_peptide / ID: 1
Details: HIV-1 envelope trimer (gp140) SpyTag conjugated to SpyCatcher mi3 nanoparticle
Enantiomer: LEVO
Source (natural)Organism: Thermotoga maritima (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: ...String:
(ASTMDAMKRGLCCVLLLCGAVFVSPSNDTWVTVYYGVPVWREAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCSNANSNTNVTDIDNSTIGEIKNCTFNVTTELRDKEKKENALFYKLDIVPLNGNNNSSFSMYRLINCNTSVVTQACPKVSFDPIPIHYCAPAGFAILKCNNKTFNGTGPCNNVSTVQCTHGIRPVVSTQLLLNGSLAEKEIIIRSENLTNNIKTIIVHLNESVEINCIRPNNNTRKSMRIGPGQTFYATGEIIGDIRQAHCNISKDKWDKTLHRVSEKLREHFPNKTITFNSSSGGDLEITTHSFNCGGEFFYCNTSGLFNSTFNTTFYEPSNLTITLQCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLILTRDGGNDNRTTETFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTNCRRRVVQGGGGSGGGGS AVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQNNLLRAPEAQQHMLQLTVWGIKQLQARVLALERYLRDQQLLGMWGCSGKLICTTNVAWNSSWSNKSQDEIWKNMTWMQWDREISDYTNTIYRLLEESQNQQEINEKDLLALDGGGGSGGGGSGGSGAHIVMVDAYKPTK)HHHHHHGSG DSATHIKFSK RDEDGKELAG ATMELRDSSG KTISTWISDG QVKDFYLYPG KYTFVETAAP DGYEVATAIT FTVNEQGQVT VNGKATKGDA HIGGSGGSGG SGGSMKMEEL FKKHKIVAVL RANSVEEAKK KALAVFLGGV HLIEITFTVP DADTVIKELS FLKEMGAIIG AGTVTSVEQA RKAVESGAEF IVSPHLDEEI SQFAKEKGVF YMPGVMTPTE LVKAMKLGHT ILKLFPGEVV GPQFVKAMKG PFPNVKFVPT GGVNLDNVCE WFKAGVLAVG VGSALVKGTP VEVAEKAKAF VEKIRGCTE

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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: 7.4
Component:
ConcentrationFormulaName
300.0 mMNaClSodium Chloride
25.0 mMC4H11NO3Tris
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 500000
Startup modelType of model: OTHER / Details: Stochastic Gradient Descent
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1439
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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