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- EMDB-44510: Cryo-EM structure of mAb8-24 bound to 426c.WITO.TM.SOSIP -

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Basic information

Entry
Database: EMDB / ID: EMD-44510
TitleCryo-EM structure of mAb8-24 bound to 426c.WITO.TM.SOSIP
Map dataSharpened map
Sample
  • Complex: Complex of 426c.WITO.TM.SOSIP bound to three mAb 8-24 Fabs
    • Protein or peptide: 426c.WITO.TM.SOSIP
    • Protein or peptide: Fv domain of heavy chain of mAb 8-24
    • Protein or peptide: Fv domain of light chain of mAb 8-24
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsHIV / antibody / IMMUNE SYSTEM
Biological speciesHomo sapiens (human) / Human immunodeficiency virus 1 / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsHurlburt NK / Pancera M
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01 AI104384 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI174242-01 United States
CitationJournal: J Virol / Year: 2024
Title: Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env.
Authors: Parul Agrawal / Maria L Knudsen / Anna MacCamy / Nicholas K Hurlburt / Arineh Khechaduri / Kelsey R Salladay / Gargi M Kher / Latha Kallur Siddaramaiah / Andrew B Stuart / Ilja Bontjer / ...Authors: Parul Agrawal / Maria L Knudsen / Anna MacCamy / Nicholas K Hurlburt / Arineh Khechaduri / Kelsey R Salladay / Gargi M Kher / Latha Kallur Siddaramaiah / Andrew B Stuart / Ilja Bontjer / Xiaoying Shen / David Montefiori / Harry B Gristick / Pamela J Bjorkman / Rogier W Sanders / Marie Pancera / Leonidas Stamatatos /
Abstract: VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and ...VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies.
IMPORTANCE: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing ...IMPORTANCE: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV. Despite an in-depth knowledge of their structures, epitopes, ontogenies, and, in a few rare cases, their maturation pathways during infection, bnAbs have, so far, not been elicited by vaccination. This necessitates the identification of key obstacles that prevent their elicitation by immunization and overcoming them. Here we examined whether CDRL1 shortening is a prerequisite for the broadly neutralizing potential of VRC01-class bnAbs, which bind within the CD4 receptor binding site of Env. Our findings indicate that CDRL1 shortening by itself is important but not sufficient for the acquisition of neutralization breadth, and suggest that particular combinations of amino acid mutations, not elicited so far by vaccination, are most likely required for the development of such a feature.
History
DepositionApr 18, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44510.png

Downloads & links

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Map

FileDownload / File: emd_44510.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 384 pix.
= 430.848 Å		1.12 Å/pix.
x 384 pix.
= 430.848 Å		1.12 Å/pix.
x 384 pix.
= 430.848 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.122 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.2593368 - 3.5536525
Average (Standard dev.)0.0010939913 (±0.0888946)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 430.848 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened Map

Fileemd_44510_additional_1.map
AnnotationUnsharpened Map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_44510_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_44510_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 : Complex of 426c.WITO.TM.SOSIP bound to three mAb 8-24 Fabs

EntireName: Complex of 426c.WITO.TM.SOSIP bound to three mAb 8-24 Fabs
Components
  • Complex: Complex of 426c.WITO.TM.SOSIP bound to three mAb 8-24 Fabs
    • Protein or peptide: 426c.WITO.TM.SOSIP
    • Protein or peptide: Fv domain of heavy chain of mAb 8-24
    • Protein or peptide: Fv domain of light chain of mAb 8-24
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Complex of 426c.WITO.TM.SOSIP bound to three mAb 8-24 Fabs

SupramoleculeName: Complex of 426c.WITO.TM.SOSIP bound to three mAb 8-24 Fabs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: 426c.WITO.TM.SOSIP

MacromoleculeName: 426c.WITO.TM.SOSIP / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 69.535422 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: AENLWVTVYY GVPVWKEAKT TLFCASDAKA YEKEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM VDQMQEDVIS IWDQSLKPC VKLTPLCVTL HCTNVTISST NGSTANVTMR EEMKNCSFNT TTVIRDKIQK EYALFYKLDI VPIEGKNTNT G YRLINCNT ...String:
AENLWVTVYY GVPVWKEAKT TLFCASDAKA YEKEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM VDQMQEDVIS IWDQSLKPC VKLTPLCVTL HCTNVTISST NGSTANVTMR EEMKNCSFNT TTVIRDKIQK EYALFYKLDI VPIEGKNTNT G YRLINCNT STCTQACPKV TFDPIPIHYC APAGYAILKC NNKTFNGKGP CNNVSTVQCT HGIKPVVSTQ LLLNGSLAEE EI VIRSKNL ADNAKIIIVQ LNKSVEIVCT RPNNNTRRSI RIGPGQTFYA TDIIGDIRQA YCNISGRNWS EAVNQVKKKL KEH FPHKNI SFQSSSGGDL EITTHSFNCG GEFFYCNTSG LFNDTISNAT IMLPCRIKQI INMWQEVGKC IYAPPIKGNI TCKS DITGL LLLRDGGNTA NNAEIFRPGG GDMRDNWRSE LYKYKVVKIE PLGVAPTRCK RRVVGRRRRR RAVGIGAVFL VFLGA AGST MGAASMTLTV QARNLLSGIV QQQSNLLRAP EAQQHLLKLT VWGIKQLQAR VLAVERYLRD QQLLGIWGCS GKLICC TNV PWNSSWSNRN LSEIWDNMTW LQWDKEISNY TQIIYGLLEE SQNQQEKNEQ DLLALD

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Macromolecule #2: Fv domain of heavy chain of mAb 8-24

MacromoleculeName: Fv domain of heavy chain of mAb 8-24 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.560518 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVQSGPE VKEPGASVRV SCKATGYTFT DHFIHWVRQA PGQGLDWMGW INPFRGGTNY PQKFQGRVTM TRDTSFTTAY MELNRLRSD DTAVYFCARG KNSDYNWDFQ HWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT ...String:
QVQLVQSGPE VKEPGASVRV SCKATGYTFT DHFIHWVRQA PGQGLDWMGW INPFRGGTNY PQKFQGRVTM TRDTSFTTAY MELNRLRSD DTAVYFCARG KNSDYNWDFQ HWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR VEPKSCDKTH HHHHH

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Macromolecule #3: Fv domain of light chain of mAb 8-24

MacromoleculeName: Fv domain of light chain of mAb 8-24 / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 22.049486 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIVMSQSPSS LAVSLGERIT LSCKSSLTLI YSYNGENYLA WYQQKPGQSP KLLIYSTSTR ESGVPDRFTG SGSGTDFTLT ISSVKAEDL AVYYCQQYEY FGGGTKLEIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
DIVMSQSPSS LAVSLGERIT LSCKSSLTLI YSYNGENYLA WYQQKPGQSP KLLIYSTSTR ESGVPDRFTG SGSGTDFTLT ISSVKAEDL AVYYCQQYEY FGGGTKLEIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV TH

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 21 / 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

Concentration1.5 mg/mL
BufferpH: 7.5
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 36000

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Image processing

Particle selectionNumber selected: 2600000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6myy

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.4.1) / Number images used: 23004
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.1)
FSC plot (resolution estimation)

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