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- EMDB-23145: Cryo-electron microscopy reconstruction of locally refined antibo... -

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Basic information

Entry
Database: EMDB / ID: EMD-23145
TitleCryo-electron microscopy reconstruction of locally refined antibody DH898.1 Fab-dimer
Map dataFinal density modified, masked, locally refined map of DH898.1 Fab dimer
Sample
  • Complex: Antibody DH898.1 Fab-dimer bound to HIV Env CH848.3.D0949.10.17chim.6R.DS.SOSIP.664 trimer
    • Complex: Antibody DH898.1 Fab-dimer
    • Complex: HIV Env CH848.3.D0949.10.17chim.6R.DS.SOSIP.664 trimer
Biological speciesMacaca mulatta (Rhesus monkey) / Human immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsEdwards RJ / Acharya P
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI140897 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI145687 United States
Citation
Journal: Cell / Year: 2021
Title: Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.
Authors: Wilton B Williams / R Ryan Meyerhoff / R J Edwards / Hui Li / Kartik Manne / Nathan I Nicely / Rory Henderson / Ye Zhou / Katarzyna Janowska / Katayoun Mansouri / Sophie Gobeil / Tyler ...Authors: Wilton B Williams / R Ryan Meyerhoff / R J Edwards / Hui Li / Kartik Manne / Nathan I Nicely / Rory Henderson / Ye Zhou / Katarzyna Janowska / Katayoun Mansouri / Sophie Gobeil / Tyler Evangelous / Bhavna Hora / Madison Berry / A Yousef Abuahmad / Jordan Sprenz / Margaret Deyton / Victoria Stalls / Megan Kopp / Allen L Hsu / Mario J Borgnia / Guillaume B E Stewart-Jones / Matthew S Lee / Naomi Bronkema / M Anthony Moody / Kevin Wiehe / Todd Bradley / S Munir Alam / Robert J Parks / Andrew Foulger / Thomas Oguin / Gregory D Sempowski / Mattia Bonsignori / Celia C LaBranche / David C Montefiori / Michael Seaman / Sampa Santra / John Perfect / Joseph R Francica / Geoffrey M Lynn / Baptiste Aussedat / William E Walkowicz / Richard Laga / Garnett Kelsoe / Kevin O Saunders / Daniela Fera / Peter D Kwong / Robert A Seder / Alberto Bartesaghi / George M Shaw / Priyamvada Acharya / Barton F Haynes /
Abstract: Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) ...Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (V) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without V-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgMIgDCD27, thus suggesting that they originated from a pool of antigen-experienced IgM or marginal zone B cells.
#1: Journal: bioRxiv / Year: 2020
Title: Fab-dimerized glycan-reactive antibodies neutralize HIV and are prevalent in humans and rhesus macaques
Authors: Williams WB / Meyerhoff RR / Edwards RJ / Li H / Nicely NI / Henderson R / Zhou Y / Janowska K / Mansouri K / Manne K / Stalls V / Hsu AL / Borgnia MJ / Stewart-Jones G / Lee MS / Bronkema N ...Authors: Williams WB / Meyerhoff RR / Edwards RJ / Li H / Nicely NI / Henderson R / Zhou Y / Janowska K / Mansouri K / Manne K / Stalls V / Hsu AL / Borgnia MJ / Stewart-Jones G / Lee MS / Bronkema N / Perfect J / Moody MA / Wiehe K / Bradley T / Kepler TB / Alam SM / Foulger A / Bonsignori M / LaBranche CC / Montefiori DC / Seaman M / Santra S / Francica JR / Lynn GM / Aussedat B / Walkowicz WE / Laga R / Kelso G / Saunders KO / Fera D / Kwong PD / Seder RA / Bartesaghi A / Shaw GM / Acharya P / Haynes BF
History
DepositionDec 17, 2020-
Header (metadata) releaseFeb 10, 2021-
Map releaseFeb 10, 2021-
UpdateJun 9, 2021-
Current statusJun 9, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.22
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.22
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7l6m
  • Surface level: 0.22
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23145.png

Downloads & links

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Map

FileDownload / File: emd_23145.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal density modified, masked, locally refined map of DH898.1 Fab dimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.023 Å		1.07 Å/pix.
x 256 pix.
= 273.023 Å		1.07 Å/pix.
x 256 pix.
= 273.023 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0665 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.22
Minimum - Maximum-0.63691163 - 0.8558629
Average (Standard dev.)0.00023892979 (±0.019697377)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.02298 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.066496093751.066496093751.06649609375
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.023273.023273.023
α/β/γ90.00090.00090.000
start NX/NY/NZ727265
NX/NY/NZ157157169
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.6370.8560.000

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Supplemental data

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Mask #1

Fileemd_23145_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_23145_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local resolution of locally refined map of DH898.1 Fab dimer

Fileemd_23145_additional_1.map
AnnotationLocal resolution of locally refined map of DH898.1 Fab dimer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unmodified locally refined map of DH898.1 Fab dimer

Fileemd_23145_additional_2.map
AnnotationUnmodified locally refined map of DH898.1 Fab dimer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A of locally refined map of DH898.1 Fab dimer

Fileemd_23145_half_map_1.map
AnnotationHalf-map A of locally refined map of DH898.1 Fab dimer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B of locally refined map of DH898.1 Fab dimer

Fileemd_23145_half_map_2.map
AnnotationHalf-map B of locally refined map of DH898.1 Fab dimer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Antibody DH898.1 Fab-dimer bound to HIV Env CH848.3.D0949.10.17ch...

EntireName: Antibody DH898.1 Fab-dimer bound to HIV Env CH848.3.D0949.10.17chim.6R.DS.SOSIP.664 trimer
Components
  • Complex: Antibody DH898.1 Fab-dimer bound to HIV Env CH848.3.D0949.10.17chim.6R.DS.SOSIP.664 trimer
    • Complex: Antibody DH898.1 Fab-dimer
    • Complex: HIV Env CH848.3.D0949.10.17chim.6R.DS.SOSIP.664 trimer

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Supramolecule #1: Antibody DH898.1 Fab-dimer bound to HIV Env CH848.3.D0949.10.17ch...

SupramoleculeName: Antibody DH898.1 Fab-dimer bound to HIV Env CH848.3.D0949.10.17chim.6R.DS.SOSIP.664 trimer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 94.2 KDa

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Supramolecule #2: Antibody DH898.1 Fab-dimer

SupramoleculeName: Antibody DH898.1 Fab-dimer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293F

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Supramolecule #3: HIV Env CH848.3.D0949.10.17chim.6R.DS.SOSIP.664 trimer

SupramoleculeName: HIV Env CH848.3.D0949.10.17chim.6R.DS.SOSIP.664 trimer
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human)

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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
Component:
ConcentrationNameFormula
5.0 mMHEPES buffer
150.0 mMsodium chlorideNaCl
GridModel: Quantifoil R1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 295 K / Instrument: LEICA EM GP
Details: 2.5 microliters of sample applied to grid and incubated for 30 seconds in chamber, blotted for 2.5 seconds, and plunge frozen in liquid ethane.
DetailsFor the DH898.1-bound complex, purified HIV-1 Env was mixed with 6-fold molar excess of recombinantly expressed and purified DH898.1 Fab to obtain a final Env concentration of 1.5 mg/ml, and incubated for 2 hours at room temperature.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 3230 / Average exposure time: 4.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1655400
Details: Particles were picked using Blob Picker on 500 micrographs with a minimum and maximum particle diameter of 150 and 250 angstroms for circular blobs, yielding 259,587 particles, which were ...Details: Particles were picked using Blob Picker on 500 micrographs with a minimum and maximum particle diameter of 150 and 250 angstroms for circular blobs, yielding 259,587 particles, which were extracted with a 320-pixel box size and subjected to 2D class averaging with 100 classes. From these, seven classes were chosen for template picking applied to the full final micrograph set, yielding 1,655,400 particles
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Startup modelType of model: INSILICO MODEL
In silico model: ab initio model generated directly from data.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.0)
Details: Local refinement of Fab-dimer bound to SOSIP trimer, after particle subtraction to remove density for SOSIP trimer, and centered on Fab-dimer.
Number images used: 59012
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15.0)
Final 3D classificationNumber classes: 2 / Avg.num./class: 31625 / Software - Name: cryoSPARC (ver. 2.15.0)
FSC plot (resolution estimation)

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