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- EMDB-18941: SARS-CoV-2 S (Spike) protein (BA.1) in complex with VHH Ma16B06 (... -

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Entry
Database: EMDB / ID: EMD-18941
TitleSARS-CoV-2 S (Spike) protein (BA.1) in complex with VHH Ma16B06 (sub-volume of two adjacent RBD-VHH modules)
Map datasharpened map
Sample
  • Complex: SARS-CoV-2 S (Spike) protein (BA.1 variant) in complex with VHH Ma16B06 (sub-volume of two adjacent RBD-VHH modules)
    • Protein or peptide: SARS-CoV-2 HexaPro S (Spike) glycoprotein (BA.1)
    • Protein or peptide: VHH antibody (nanobody) Ma16B06
KeywordsSARS-CoV-2 / Spike Glycoprotein / VHH Antibody (Nanobody) Complex / Inhibitor / VIRAL PROTEIN
Biological speciesSevere acute respiratory syndrome coronavirus / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsGuttler T / Aksu M / Gorlich D
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Antiviral Res / Year: 2024
Title: Nanobodies to multiple spike variants and inhalation of nanobody-containing aerosols neutralize SARS-CoV-2 in cell culture and hamsters.
Authors: Metin Aksu / Priya Kumar / Thomas Güttler / Waltraud Taxer / Kathrin Gregor / Bianka Mußil / Oleh Rymarenko / Kim M Stegmann / Antje Dickmanns / Sabrina Gerber / Wencke Reineking / Claudia ...Authors: Metin Aksu / Priya Kumar / Thomas Güttler / Waltraud Taxer / Kathrin Gregor / Bianka Mußil / Oleh Rymarenko / Kim M Stegmann / Antje Dickmanns / Sabrina Gerber / Wencke Reineking / Claudia Schulz / Timo Henneck / Ahmed Mohamed / Gerhard Pohlmann / Mehmet Ramazanoglu / Kemal Mese / Uwe Groß / Tamar Ben-Yedidia / Oded Ovadia / Dalit Weinstein Fischer / Merav Kamensky / Amir Reichman / Wolfgang Baumgärtner / Maren von Köckritz-Blickwede / Matthias Dobbelstein / Dirk Görlich /
Abstract: The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single- ...The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.
History
DepositionNov 18, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18941.png

Downloads & links

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Map

FileDownload / File: emd_18941.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 224 pix.
= 235.2 Å		1.05 Å/pix.
x 224 pix.
= 235.2 Å		1.05 Å/pix.
x 224 pix.
= 235.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.033
Minimum - Maximum-0.039773658 - 0.11497164
Average (Standard dev.)-0.00020236326 (±0.003282721)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 235.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18941_msk_1.map
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Additional map: unsharpened map

Fileemd_18941_additional_1.map
Annotationunsharpened map
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Half map: half map 2

Fileemd_18941_half_map_1.map
Annotationhalf map 2
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Half map: half map 1

Fileemd_18941_half_map_2.map
Annotationhalf map 1
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Sample components

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Entire : SARS-CoV-2 S (Spike) protein (BA.1 variant) in complex with VHH M...

EntireName: SARS-CoV-2 S (Spike) protein (BA.1 variant) in complex with VHH Ma16B06 (sub-volume of two adjacent RBD-VHH modules)
Components
  • Complex: SARS-CoV-2 S (Spike) protein (BA.1 variant) in complex with VHH Ma16B06 (sub-volume of two adjacent RBD-VHH modules)
    • Protein or peptide: SARS-CoV-2 HexaPro S (Spike) glycoprotein (BA.1)
    • Protein or peptide: VHH antibody (nanobody) Ma16B06

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Supramolecule #1: SARS-CoV-2 S (Spike) protein (BA.1 variant) in complex with VHH M...

SupramoleculeName: SARS-CoV-2 S (Spike) protein (BA.1 variant) in complex with VHH Ma16B06 (sub-volume of two adjacent RBD-VHH modules)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Trimeric SARS-CoV-2 S (Spike) protein (BA.1 variant) in complex with VHH antibody (nanobody) Ma16B06 (one copy per protomer).
Source (natural)Organism: Severe acute respiratory syndrome coronavirus / Strain: BA.1
Molecular weightTheoretical: 600 KDa

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Macromolecule #1: SARS-CoV-2 HexaPro S (Spike) glycoprotein (BA.1)

MacromoleculeName: SARS-CoV-2 HexaPro S (Spike) glycoprotein (BA.1) / type: protein_or_peptide / ID: 1
Details: The Spike protein is a trimeric assembly. The map only shows two adjacent receptor-binding domains (RBDs), associated with one VHH antibody (Ma16B06) each.
Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus / Strain: BA.1
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VNLTTRTQLP PAYTNSFTRG VYYPDKVFRS SVLHSTQDLF LPFFSNVTWF HVISGTNGTK RFDNPVLPFN DGVYFASIEK SNIIRGWIFG TTLDSKTQSL LIVNNATNVV IKVCEFQFCN DPFLDHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR ...String:
VNLTTRTQLP PAYTNSFTRG VYYPDKVFRS SVLHSTQDLF LPFFSNVTWF HVISGTNGTK RFDNPVLPFN DGVYFASIEK SNIIRGWIFG TTLDSKTQSL LIVNNATNVV IKVCEFQFCN DPFLDHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI IVREPEDLPQ GFSALEPLVD LPIGINITRF QTLLALHRSY LTPGDSSSGW TAGAAAYYVG YLQPRTFLLK YNENGTITDA VDCALDPLSE TKCTLKSFTV EKGIYQTSNF RVQPTESIVR FPNITNLCPF DEVFNATRFA SVYAWNRKRI SNCVADYSVL YNLAPFFTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGNI ADYNYKLPDD FTGCVIAWNS NKLDSKVSGN YNYLYRLFRK SNLKPFERDI STEIYQAGNK PCNGVAGFNC YFPLRSYSFR PTYGVGHQPY RVVVLSFELL HAPATVCGPK KSTNLVKNKC VNFNFNGLKG TGVLTESNKK FLPFQQFGRD IADTTDAVRD PQTLEILDIT PCSFGGVSVI TPGTNTSNQV AVLYQGVNCT EVPVAIHADQ LTPTWRVYST GSNVFQTRAG CLIGAEYVNN SYECDIPIGA GICASYQTQT KSPGSASSVA SQSIIAYTMS LGAENSVAYS NNSIAIPTNF TISVTTEILP VSMTKTSVDC TMYICGDSTE CSNLLLQYGS FCTQLKRALT GIAVEQDKNT QEVFAQVKQI YKTPPIKYFG GFNFSQILPD PSKPSKRSPI EDLLFNKVTL ADAGFIKQYG DCLGDIAARD LICAQKFKGL TVLPPLLTDE MIAQYTSALL AGTITSGWTF GAGPALQIPF PMQMAYRFNG IGVTQNVLYE NQKLIANQFN SAIGKIQDSL SSTPSALGKL QDVVNHNAQA LNTLVKQLSS KFGAISSVLN DIFSRLDPPE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQSAPH GVVFLHVTYV PAQEKNFTTA PAICHDGKAH FPREGVFVSN GTHWFVTQRN FYEPQIITTD NTFVSGNCDV VIGIVNNTVY DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN IQKEIDRLNE VAKNLNESLI DLQELGKYEQ GSGYIPEAPR DGQAYVRKDG EWVLLSTFLG RSLEVLFQGP GHHHHHHHHS AWSHPQFEKG GGSGGGGSGG SAWSHPQFEK

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Macromolecule #2: VHH antibody (nanobody) Ma16B06

MacromoleculeName: VHH antibody (nanobody) Ma16B06 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSQVQLVESG GGLVRTGGSL RLSCAASGSI LQIWAMKWYR QAPGLQREWI ATIPNSGEPF YASSVEGRFT GSRENEETVY LYLNNLEPED TAVYYCEVNE GVPVREYWGQ GTQVTVSSTS

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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

Concentration2.0 mg/mL
BufferpH: 8
Component:
ConcentrationName
2.0 mMTris/HCl pH 8.0
150.0 mMNaCl
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailsmonodisperse material eluted from a size exclusion column (Superose 6 Increase 3.2/300, Cytiva)

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7592 / Average exposure time: 0.075 sec. / Average electron dose: 39.67 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
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

DetailsWarp 1.0.9 (Tegunov and Cramer, 2019) was used (in parallel to data acquisition) for motion correction, dose weighting and CTF estimation.
Particle selectionNumber selected: 3510830
Details: Warp 1.0.9 (Tegunov and Cramer, 2019) was used (in parallel to data acquisition) for particle selection.
Startup modelType of model: EMDB MAP
EMDB ID:

13106

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 219553
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 79600 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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