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- EMDB-23565: Cryo-EM structure of EDC-crosslinked ConSOSL.UFO.664 (ConS-EDC) i... -

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Basic information

Entry
Database: EMDB / ID: EMD-23565
TitleCryo-EM structure of EDC-crosslinked ConSOSL.UFO.664 (ConS-EDC) in complex with bNAb PGT122
Map datafiltered and B-factor sharpened map
Sample
  • Complex: EDC-crosslinked ConSOSL.UFO.664 in complex with bNAb PGT122
    • Complex: EDC-crosslinked ConSOSL.UFO.664
      • Protein or peptide: Env glycoprotein gp160
    • Complex: PGT122 Fab
      • Protein or peptide: PGT122 Fab light chain
      • Protein or peptide: PGT122 Fab heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsEnv / SOSIP / VIRAL PROTEIN / VIRAL PROTEIN-IMMUNE SYSTEM complex
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsMartin GM / Ward AB
Funding support1 items
OrganizationGrant numberCountry
Bill & Melinda Gates Foundation
CitationJournal: NPJ Vaccines / Year: 2023
Title: Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens.
Authors: Gregory M Martin / Rebecca A Russell / Philip Mundsperger / Scarlett Harris / Lu Jovanoska / Luiza Farache Trajano / Torben Schiffner / Katalin Fabian / Monica Tolazzi / Gabriella Scarlatti ...Authors: Gregory M Martin / Rebecca A Russell / Philip Mundsperger / Scarlett Harris / Lu Jovanoska / Luiza Farache Trajano / Torben Schiffner / Katalin Fabian / Monica Tolazzi / Gabriella Scarlatti / Leon McFarlane / Hannah Cheeseman / Yoann Aldon / Edith E Schermer / Marielle Breemen / Kwinten Sliepen / Dietmar Katinger / Renate Kunert / Rogier W Sanders / Robin Shattock / Andrew B Ward / Quentin J Sattentau /
Abstract: Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local ...Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4-0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design.
History
DepositionMar 3, 2021-
Header (metadata) releaseMar 9, 2022-
Map releaseMar 9, 2022-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7lx3
  • Surface level: 0.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23565.png

Downloads & links

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Map

FileDownload / File: emd_23565.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfiltered and B-factor sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 220 pix.
= 226.6 Å		1.03 Å/pix.
x 220 pix.
= 226.6 Å		1.03 Å/pix.
x 220 pix.
= 226.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.4531895 - 3.060852
Average (Standard dev.)0.004455128 (±0.14411995)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 226.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z226.600226.600226.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-1.4533.0610.004

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Supplemental data

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Sample components

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Entire : EDC-crosslinked ConSOSL.UFO.664 in complex with bNAb PGT122

EntireName: EDC-crosslinked ConSOSL.UFO.664 in complex with bNAb PGT122
Components
  • Complex: EDC-crosslinked ConSOSL.UFO.664 in complex with bNAb PGT122
    • Complex: EDC-crosslinked ConSOSL.UFO.664
      • Protein or peptide: Env glycoprotein gp160
    • Complex: PGT122 Fab
      • Protein or peptide: PGT122 Fab light chain
      • Protein or peptide: PGT122 Fab heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: EDC-crosslinked ConSOSL.UFO.664 in complex with bNAb PGT122

SupramoleculeName: EDC-crosslinked ConSOSL.UFO.664 in complex with bNAb PGT122
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: EDC-crosslinked ConSOSL.UFO.664

SupramoleculeName: EDC-crosslinked ConSOSL.UFO.664 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Human immunodeficiency virus 1

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Supramolecule #3: PGT122 Fab

SupramoleculeName: PGT122 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Env glycoprotein gp160

MacromoleculeName: Env glycoprotein gp160 / type: protein_or_peptide / ID: 1 / Details: ConSOSL.UFO.664 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 73.136742 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: MDAMKRGLCC VLLLCGAVFV SPSQEIHARF RRGARSRVEN LWVTVYYGVP VWKDAETTLF CASDAKAYDT EKRNVWATHA CVPTDPNPQ EIVLENVTEN FNMWKNNMVE QMHTDIISLW DQSLKPCVKL TPLCVTLNCT NVNVTNTTNN TEEKGEIKNC S FNITTELR ...String:
MDAMKRGLCC VLLLCGAVFV SPSQEIHARF RRGARSRVEN LWVTVYYGVP VWKDAETTLF CASDAKAYDT EKRNVWATHA CVPTDPNPQ EIVLENVTEN FNMWKNNMVE QMHTDIISLW DQSLKPCVKL TPLCVTLNCT NVNVTNTTNN TEEKGEIKNC S FNITTELR DKKKKVYALF YRLDVVPIDD NNNNSSNYRL INCNTSAITQ ACPKVSFEPI PIHYCAPAGF AILKCNDKKF NG TGPCKNV STVQCTHGIK PVVSTQLLLN GSLAEEEIII RSENITNNAK TIIVQLNESV EINCTRPNNN TRKSIRIGPG QWF YATGDI IGDIRQAHCN ISGTKWNKTL QQVVKKLREH FNNKTIIFNP SSGGDLEITT HSFNCGGEFF YCNTSGLFNS TWIG NGTKN NNNTNDTITL PCRIKQIINM WQRVGQPMYA PPIQGKIRCV SNITGLLLTR DGGNNNTNET ETFRPGGGDM RDNWR SELY KYKVVKIEPL GVAPTRCKRR VVEGGGGGSG GGGSAVGIGA VFLGFLGAAG STMGAASMTL TVQARNLLSG GSGSGS GST VWGIKQLQAR VLAVERYLRD QQLLGIWGCS GKLICCTNVP WNSSWSNKSQ DEIWDNMTWM EWDKEINNYT DIIYSLI EE SQNQQEKNEQ DLLALD

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Macromolecule #2: PGT122 Fab light chain

MacromoleculeName: PGT122 Fab light chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.880275 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: APTFVSVAPG QTARITCGEE SLGSRSVIWY QQRPGQAPSL IIYNNNDRPS GIPDRFSGSP GSTFGTTATL TITSVEAGDE ADYYCHIWD SRRPTNWVFG EGTTLIVLSQ PKAAPSVTLF PPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAG V ETTTPSKQ ...String:
APTFVSVAPG QTARITCGEE SLGSRSVIWY QQRPGQAPSL IIYNNNDRPS GIPDRFSGSP GSTFGTTATL TITSVEAGDE ADYYCHIWD SRRPTNWVFG EGTTLIVLSQ PKAAPSVTLF PPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAG V ETTTPSKQ SNNKYAASSY LSLTPEQWKS HKSYSCQVTH EGSTVEKTVA PTECS

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Macromolecule #3: PGT122 Fab heavy chain

MacromoleculeName: PGT122 Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.434691 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QVHLQESGPG LVKPSETLSL TCNVSGTLVR DNYWSWIRQP LGKQPEWIGY VHDSGDTNYN PSLKSRVHLS LDKSKNLVSL RLTGVTAAD SAIYYCATTK HGRRIYGVVA FKEWFTYFYM DVWGKGTSVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP ...String:
QVHLQESGPG LVKPSETLSL TCNVSGTLVR DNYWSWIRQP LGKQPEWIGY VHDSGDTNYN PSLKSRVHLS LDKSKNLVSL RLTGVTAAD SAIYYCATTK HGRRIYGVVA FKEWFTYFYM DVWGKGTSVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSC

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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: 15 / 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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 132156
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING

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