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- EMDB-27693: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex wi... -

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Entry
Database: EMDB / ID: EMD-27693
TitleLM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein (global refinement)
Map dataSharpened map
Sample
  • Complex: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein
Keywordsnanobody / bispecific nanobody / coronavirus / antibody engineering / VIRAL PROTEIN
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsOzorowski G / Turner HL / Ward AB
Funding support United States, 2 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1170236 United States
Bill & Melinda Gates FoundationINV-004923 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.
Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan ...Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan Verma / Jingjia Liu / Robyn L Stanfield / Xueyong Zhu / Hannah L Turner / Devin Sok / Po-Ssu Huang / Dennis R Burton / Andrew B Ward / Ian A Wilson / Joseph G Jardine /
Abstract: Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple ...Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human V3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the V and V domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.
History
DepositionJul 25, 2022-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27693.png

Downloads & links

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Map

FileDownload / File: emd_27693.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 380 pix.
= 397.1 Å		1.05 Å/pix.
x 380 pix.
= 397.1 Å		1.05 Å/pix.
x 380 pix.
= 397.1 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.045 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.65071166 - 1.5290818
Average (Standard dev.)-0.0012748191 (±0.036684144)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 397.09998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27693_msk_1.map
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Additional map: unsharpened map

Fileemd_27693_additional_1.map
Annotationunsharpened map
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Half map: Half map 1

Fileemd_27693_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_27693_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex wi...

EntireName: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein
Components
  • Complex: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein

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Supramolecule #1: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex wi...

SupramoleculeName: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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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

Concentration0.85 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
50.0 mMTris
150.0 mMsodium chlorideNaCl
0.06 mMn-Dodecyl-B-D-Maltopyranoside

Details: Detergent added shortly before grid application
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailsincubated complex for 100 minutes at room temperature prior to freezing

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 4296 / Average exposure time: 9.0 sec. / Average electron dose: 49.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: cryosparc ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 61539
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL

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