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- EMDB-21231: De novo designed tetrahedral nanoparticle T33_dn2 presenting BG50... -

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Basic information

Entry
Database: EMDB / ID: EMD-21231
TitleDe novo designed tetrahedral nanoparticle T33_dn2 presenting BG505 SOSIP trimers
Map dataDe novo designed tetrahedral nanoparticle T33_dn2 presenting BG505 SOSIP trimers, post-processed cryoEM map
Sample
  • Complex: De novo designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
    • Protein or peptide: T33_dn2A
    • Protein or peptide: T33_dn2B
KeywordsHIV Env / de novo / nanoparticles / vaccine design / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsWard AB / Antanasijevic A
Funding support United States, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782 United States
CitationJournal: NPJ Vaccines / Year: 2020
Title: Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.
Authors: Jacob T Martin / Christopher A Cottrell / Aleksandar Antanasijevic / Diane G Carnathan / Benjamin J Cossette / Chiamaka A Enemuo / Etse H Gebru / Yury Choe / Federico Viviano / Stephanie ...Authors: Jacob T Martin / Christopher A Cottrell / Aleksandar Antanasijevic / Diane G Carnathan / Benjamin J Cossette / Chiamaka A Enemuo / Etse H Gebru / Yury Choe / Federico Viviano / Stephanie Fischinger / Talar Tokatlian / Kimberly M Cirelli / George Ueda / Jeffrey Copps / Torben Schiffner / Sergey Menis / Galit Alter / William R Schief / Shane Crotty / Neil P King / David Baker / Guido Silvestri / Andrew B Ward / Darrell J Irvine /
Abstract: Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic ...Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
History
DepositionJan 22, 2020-
Header (metadata) releaseFeb 19, 2020-
Map releaseAug 12, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vl6
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21231.png

Downloads & links

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Map

FileDownload / File: emd_21231.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDe novo designed tetrahedral nanoparticle T33_dn2 presenting BG505 SOSIP trimers, post-processed cryoEM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 440 pix.
= 506. Å		1.15 Å/pix.
x 440 pix.
= 506. Å		1.15 Å/pix.
x 440 pix.
= 506. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.02345367 - 0.073088616
Average (Standard dev.)0.00017986611 (±0.0020430344)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 506.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z506.000506.000506.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS440440440
D min/max/mean-0.0230.0730.000

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Supplemental data

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Mask #1

Fileemd_21231_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: De novo designed tetrahedral nanoparticle T33 dn2 presenting BG505...

Fileemd_21231_half_map_1.map
AnnotationDe novo designed tetrahedral nanoparticle T33_dn2 presenting BG505 SOSIP trimers, cryoEM half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: De novo designed tetrahedral nanoparticle T33 dn2 presenting BG505...

Fileemd_21231_half_map_2.map
AnnotationDe novo designed tetrahedral nanoparticle T33_dn2 presenting BG505 SOSIP trimers, cryoEM half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : De novo designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2

EntireName: De novo designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
Components
  • Complex: De novo designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
    • Protein or peptide: T33_dn2A
    • Protein or peptide: T33_dn2B

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Supramolecule #1: De novo designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2

SupramoleculeName: De novo designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: De novo designed T33_dn2 nanoparticle was engineered to present BG505 SOSIP trimers. 4 trimers per nanoparticle.
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: T33_dn2A

MacromoleculeName: T33_dn2A / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.136632 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
GSMGNLAEKM YKAGNAMYRK GQYTIAIIAY TLALLKDPNN AEAWYNLGNA AYKKGEYDEA IEAYQKALEL DPNNAEAWYN LGNAYYKQG DYDEAIEYYK KALRLDPRNV DAIENLIEAE EKQGAS

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Macromolecule #2: T33_dn2B

MacromoleculeName: T33_dn2B / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.905603 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MEEAELAYLL GELAYKLGEY RIAIRAYRIA LKRDPNNAEA WYNLGNAYYK QGDYREAIRY YLRALKLDPE NAEAWYNLGN ALYKQGKYD LAIIAYQAAL EEDPNNAEAK QNLGNAKQKQ GLEHHHHHH

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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

Concentration4.0 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
25.0 mMTris-HCl
150.0 mMsodium chlorideNaCl

Details: Freshly prepared buffer, 0.2 uM filtered
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 10 K / Instrument: FEI VITROBOT MARK IV
Details: Lauryl maltose neopentyl glycol (LMNG) at a final concentration of 0.005 mM was added to the nanoparticle sample (4.0 mg/mL) and 3 uL was immediately loaded onto plasma-cleaned Quantifoil R ...Details: Lauryl maltose neopentyl glycol (LMNG) at a final concentration of 0.005 mM was added to the nanoparticle sample (4.0 mg/mL) and 3 uL was immediately loaded onto plasma-cleaned Quantifoil R 2/1 holey carbon copper grid (Cu, 400-mesh, Quantifoil Micro Tools GmbH)..
DetailsBG505 SOSIP-T33_dn2 nanoparticles were prepared by combining equimolar amounts of BG505 SOSIP-T33_dn2A and T33_dn2B components and subsequent incubation.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 3 / Number real images: 2748 / Average exposure time: 11.25 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 210597 / Details: Template picker in cryoSPARC
Startup modelType of model: OTHER / Details: Low resolution T33_dn2 nanoparticle map
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: T (tetrahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 35521
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5cez


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6vl6:
De novo designed tetrahedral nanoparticle T33_dn2 presenting BG505 SOSIP trimers

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