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- EMDB-41025: MD65 N332-GT5 SOSIP in complex with RM_N332_36 Fab and RM20A3 Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-41025
TitleMD65 N332-GT5 SOSIP in complex with RM_N332_36 Fab and RM20A3 Fab
Map datasharpened map
Sample
  • Complex: MD65 N332-GT5 SOSIP in complex with RM_N332_36 Fab and RM20A3 Fab
    • Protein or peptide: MD65 N332-GT5 SOSIP gp120
    • Protein or peptide: RM20A3 heavy chain Fv
    • Protein or peptide: RM20A3 light chain Fv
    • Protein or peptide: MD65 N332-GT5 SOSIP gp41
    • Protein or peptide: RM_N332_36 heavy chain Fv
    • Protein or peptide: RM_N332_36 light chain Fv
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsgermline targeting / NHP / SOSIP / Env / HIV / BG18 / immunogen design / antibody / VIRAL PROTEIN / VIRAL PROTEIN-Immune System complex
Biological speciesMacaca mulatta (Rhesus monkey) / Human immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsOzorowski G / Torres JL / Ward AB
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 Al100663 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 AI144462 United States
CitationJournal: Science / Year: 2024
Title: Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.
Authors: Jon M Steichen / Ivy Phung / Eugenia Salcedo / Gabriel Ozorowski / Jordan R Willis / Sabyasachi Baboo / Alessia Liguori / Christopher A Cottrell / Jonathan L Torres / Patrick J Madden / ...Authors: Jon M Steichen / Ivy Phung / Eugenia Salcedo / Gabriel Ozorowski / Jordan R Willis / Sabyasachi Baboo / Alessia Liguori / Christopher A Cottrell / Jonathan L Torres / Patrick J Madden / Krystal M Ma / Henry J Sutton / Jeong Hyun Lee / Oleksandr Kalyuzhniy / Joel D Allen / Oscar L Rodriguez / Yumiko Adachi / Tina-Marie Mullen / Erik Georgeson / Michael Kubitz / Alison Burns / Shawn Barman / Rohini Mopuri / Amanda Metz / Tasha K Altheide / Jolene K Diedrich / Swati Saha / Kaitlyn Shields / Steven E Schultze / Melissa L Smith / Torben Schiffner / Dennis R Burton / Corey T Watson / Steven E Bosinger / Max Crispin / John R Yates / James C Paulson / Andrew B Ward / Devin Sok / Shane Crotty / William R Schief /
Abstract: Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically ...Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.
History
DepositionJun 8, 2023-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41025.png

Downloads & links

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Map

FileDownload / File: emd_41025.map.gz / Format: CCP4 / Size: 274.6 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.)
1.15 Å/pix.
x 416 pix.
= 478.4 Å		1.15 Å/pix.
x 416 pix.
= 478.4 Å		1.15 Å/pix.
x 416 pix.
= 478.4 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: 0.28
Minimum - Maximum-0.6305448 - 1.6800833
Average (Standard dev.)-0.00031379797 (±0.03235471)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 478.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41025_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_41025_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_41025_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MD65 N332-GT5 SOSIP in complex with RM_N332_36 Fab and RM20A3 Fab

EntireName: MD65 N332-GT5 SOSIP in complex with RM_N332_36 Fab and RM20A3 Fab
Components
  • Complex: MD65 N332-GT5 SOSIP in complex with RM_N332_36 Fab and RM20A3 Fab
    • Protein or peptide: MD65 N332-GT5 SOSIP gp120
    • Protein or peptide: RM20A3 heavy chain Fv
    • Protein or peptide: RM20A3 light chain Fv
    • Protein or peptide: MD65 N332-GT5 SOSIP gp41
    • Protein or peptide: RM_N332_36 heavy chain Fv
    • Protein or peptide: RM_N332_36 light chain Fv
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: MD65 N332-GT5 SOSIP in complex with RM_N332_36 Fab and RM20A3 Fab

SupramoleculeName: MD65 N332-GT5 SOSIP in complex with RM_N332_36 Fab and RM20A3 Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Macaca mulatta (Rhesus monkey)

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Macromolecule #1: MD65 N332-GT5 SOSIP gp120

MacromoleculeName: MD65 N332-GT5 SOSIP gp120 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 54.286773 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: AENLWVTVYY GVPVWKDAET TLFCASDAKA YETEKHNVWA THACVPTDPN PQEIHLENVT EEFNMWKNNM VEQMHEDIIS LWDQSLKPC VKLTPLCVTL QCTNYAPKLR SMMRGEIKNC SFNMTTELRD KKQKVYSLFY RLDVVQINEN QGNRSNNSNK E YRLINCNT ...String:
AENLWVTVYY GVPVWKDAET TLFCASDAKA YETEKHNVWA THACVPTDPN PQEIHLENVT EEFNMWKNNM VEQMHEDIIS LWDQSLKPC VKLTPLCVTL QCTNYAPKLR SMMRGEIKNC SFNMTTELRD KKQKVYSLFY RLDVVQINEN QGNRSNNSNK E YRLINCNT SAITQACPKV SFEPIPIHYC APAGFAILKC KDKKFNGTGP CQNVSTVQCT HGIKPVVSTQ LLLNGSLAEE EV IIRSENI TNNAKNILVQ LNTSVQINCT RPSNNTVKSI RIGPGQAFYY FGDVLGHVRM AHCNISKATW NETLGKVVKQ LRK HFGNNT IIRFAQSSGG DLEVTTHSFN CGGEFFYCNT SGLFNSTWIS NTSVQGSNST GSNDSLILPC WIKQIINMWQ RIGQ AMYAP PIQGVIRCVS NITGLILTRD GGSTNSTTET FRPGGGDMRD NWRSELYKYK VVKIEPLGVA PTRCKRRTVG RRRRR R

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Macromolecule #2: RM20A3 heavy chain Fv

MacromoleculeName: RM20A3 heavy chain Fv / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 13.511111 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVQLVETGGG LVQPGGSLKL SCRASGYTFS SFAMSWVRQA PGKGLEWVSL INDRGGLTFY VDSVKGRFTI SRDNSKNTLS LQMHSLRDG DTAVYYCATG GMSSALQSSK YYFDFWGQGA LVTVSS

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Macromolecule #3: RM20A3 light chain Fv

MacromoleculeName: RM20A3 light chain Fv / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 13.5088 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
ALTQPPSVSG SPGQSVTISC TGTSSDIGSY NYVSWYQQHP GKAPKLMIYD VTQRPSGVSD RFSGSKSGNT ASLTISGLQA DDEADYYCS AYAGRQTFYI FGGGTRLTVL GQPKASPTVT LFPPSSEEL

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Macromolecule #4: MD65 N332-GT5 SOSIP gp41

MacromoleculeName: MD65 N332-GT5 SOSIP gp41 / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 17.096148 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
AAGIGASSDG FLGAAGSTMG AASMTLTVQA RNLLSGIVQQ QSNLLRAPEP QQHLLKDTHW GIKQLQARVL AVEHYLRDQQ LLGIWGCSG KLICCTNVPW NSSWSNRNLS EIWDNMTWLQ WDKEISNYTQ IIYGLLEESQ NQQEKNEQDL LALD

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Macromolecule #5: RM_N332_36 heavy chain Fv

MacromoleculeName: RM_N332_36 heavy chain Fv / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 14.779726 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QVTLKESGPA LVKPTQTLTL TCTFSGFSLS TNYMGVDWIR QPPGKALEWL ARIYWNDDKY YSTSLKSRLT ISKDTSKNQV VLTMSNMDP VDTATYYCAR VKLSVWGVLQ FFEWSDYGLD SWGQGVVVTV SS

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Macromolecule #6: RM_N332_36 light chain Fv

MacromoleculeName: RM_N332_36 light chain Fv / type: protein_or_peptide / ID: 6 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 11.40742 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
SYVLTQPPSV SVSPGQTARI TCGADNIGSQ SVHWYQQKSP QAPVLVISAD TERPSGIPER FSGSNSGNTA TLTISGVEAG DEADYYCQV WDSDSKYALF GGGTRLTVL

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Macromolecule #9: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 9 / Number of copies: 51 / 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

Concentration6 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 51.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: ab initio
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 75027
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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