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- EMDB-21230: BG505 SOSIP reconstructed from a designed tetrahedral nanoparticl... -

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Entry
Database: EMDB / ID: EMD-21230
TitleBG505 SOSIP reconstructed from a designed tetrahedral nanoparticle, BG505 SOSIP-T33_dn2
Map dataBG505 SOSIP reconstructed from a BG505 SOSIP-T33_dn2 nanoparticle, main post-processed cryo-EM map
Sample
  • Complex: BG505 SOSIP trimer reconstructed from a designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
    • Protein or peptide: BG505 SOSIP.v5.2(7S) - gp120
    • Protein or peptide: BG505 SOSIP.v5.2(7S) - gp41
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 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-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6vl5
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21230.png

Downloads & links

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Map

FileDownload / File: emd_21230.map.gz / Format: CCP4 / Size: 26.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBG505 SOSIP reconstructed from a BG505 SOSIP-T33_dn2 nanoparticle, main post-processed cryo-EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 190 pix.
= 218.5 Å		1.15 Å/pix.
x 190 pix.
= 218.5 Å		1.15 Å/pix.
x 190 pix.
= 218.5 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: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.06283325 - 0.09198444
Average (Standard dev.)0.0011087788 (±0.0054309373)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions190190190
Spacing190190190
CellA=B=C: 218.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z190190190
origin x/y/z0.0000.0000.000
length x/y/z218.500218.500218.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS190190190
D min/max/mean-0.0630.0920.001

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Supplemental data

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Mask #1

Fileemd_21230_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BG505 SOSIP reconstructed from a BG505 SOSIP-T33 dn2 nanoparticle,...

Fileemd_21230_half_map_1.map
AnnotationBG505 SOSIP reconstructed from a BG505 SOSIP-T33_dn2 nanoparticle, cryo-EM half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BG505 SOSIP reconstructed from a BG505 SOSIP-T33 dn2 nanoparticle,...

Fileemd_21230_half_map_2.map
AnnotationBG505 SOSIP reconstructed from a BG505 SOSIP-T33_dn2 nanoparticle, cryo-EM 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 : BG505 SOSIP trimer reconstructed from a designed tetrahedral nano...

EntireName: BG505 SOSIP trimer reconstructed from a designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
Components
  • Complex: BG505 SOSIP trimer reconstructed from a designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
    • Protein or peptide: BG505 SOSIP.v5.2(7S) - gp120
    • Protein or peptide: BG505 SOSIP.v5.2(7S) - gp41
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: BG505 SOSIP trimer reconstructed from a designed tetrahedral nano...

SupramoleculeName: BG505 SOSIP trimer reconstructed from a designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: BG505 SOSIP trimers were extracted from the T33_dn2 nanoparticles and reconstructed as subparticles.
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293F

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Macromolecule #1: BG505 SOSIP.v5.2(7S) - gp120

MacromoleculeName: BG505 SOSIP.v5.2(7S) - gp120 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 56.806613 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLE NVTEEFNMWK NNMVEQMHTD IISLWDQSLK PCVKLTPLCV TLQCTNVTNN ITDDMRGELK NCSFNMTTEL R DKKQKVYS ...String:
MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLE NVTEEFNMWK NNMVEQMHTD IISLWDQSLK PCVKLTPLCV TLQCTNVTNN ITDDMRGELK NCSFNMTTEL R DKKQKVYS LFYRLDVVQI NENQGNRSNN SNKEYRLINC NTSAITQACP KVSFEPIPIH YCAPAGFAIL KCKDKKFNGT GP CTNVSTV QCTHGIKPVV STQLLLNGSL AEEEVIIRSE NITNNAKNIL VQLNESVQIN CTRPNNNTVK SIRIGPGQWF YYT GDIIGD IRQAHCNVSK ATWNETLGKV VKQLRKHFGN NTIIRFANSS GGDLEVTTHS FNCGGEFFYC NTSGLFNSTW ISNT SVQGS NSTGSNDSIT LPCRIKQIIN MWQRIGQAMY APPIQGVIRC VSNITGLILT RDGGSTNSTT ETFRPGGGDM RDNWR SELY KYKVVKIEPL GVAPTRCKRR VV

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Macromolecule #2: BG505 SOSIP.v5.2(7S) - gp41

MacromoleculeName: BG505 SOSIP.v5.2(7S) - gp41 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.971518 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
GRRRRRRAVG IGAVSLGFLG AAGSTMGAAS MTLTVQARNL LSGIVQQQSN LLRAPECQQH LLKDTHWGIK QLQARVLAVE HYLRDQQLL GIWGCSGKLI CCTNVPWNSS WSNRNLSEIW DNMTWLQWDK EISNYTQIIY GLLEESQNQQ EKNEQDLLEL D KWASLW

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 36 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
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: 142084
Details: Starting number of BG505 SOSIP-T33_dn2 nanoparticles was 35,521. BG505 SOSIP subparticles were extracted using LocalRec v1.2.0. There are 4 trimeric BG505 SOSIP subparticles per one T33_dn2 ...Details: Starting number of BG505 SOSIP-T33_dn2 nanoparticles was 35,521. BG505 SOSIP subparticles were extracted using LocalRec v1.2.0. There are 4 trimeric BG505 SOSIP subparticles per one T33_dn2 nanoparticle, making a total of 142,084 starting subparticles.
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: Low resolution BG505 SOSIP trimer map
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 52939
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

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6vl5:
BG505 SOSIP reconstructed from a designed tetrahedral nanoparticle, BG505 SOSIP-T33_dn2

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