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- EMDB-19068: CryoEM reconstruction of SARS-CoV-2 Spike in complex with nanobod... -

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Basic information

Entry
Database: EMDB / ID: EMD-19068
TitleCryoEM reconstruction of SARS-CoV-2 Spike in complex with nanobody tri-TMH (closed conformation)
Map dataMap filtered to local resolution
Sample
  • Complex: SARS-CoV-2 spike in complex with nanobody tri-TMH
    • Protein or peptide: SARS-CoV-2 Spike protein
    • Protein or peptide: Nanobody tri-TMH
KeywordsSARS-CoV-2 spike / nanobody / trimodular nanobody / VIRAL PROTEIN
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.48 Å
AuthorsRissanen I / Hannula L / Huiskonen JT
Funding support Finland, 3 items
OrganizationGrant numberCountry
Academy of Finland336492 Finland
Academy of Finland342988 Finland
Academy of Finland346508 Finland
CitationJournal: Microbiol Spectr / Year: 2024
Title: Nanobody engineering for SARS-CoV-2 neutralization and detection.
Authors: Liina Hannula / Suvi Kuivanen / Jonathan Lasham / Ravi Kant / Lauri Kareinen / Mariia Bogacheva / Tomas Strandin / Tarja Sironen / Jussi Hepojoki / Vivek Sharma / Petri Saviranta / Anja ...Authors: Liina Hannula / Suvi Kuivanen / Jonathan Lasham / Ravi Kant / Lauri Kareinen / Mariia Bogacheva / Tomas Strandin / Tarja Sironen / Jussi Hepojoki / Vivek Sharma / Petri Saviranta / Anja Kipar / Olli Vapalahti / Juha T Huiskonen / Ilona Rissanen /
Abstract: In response to the ongoing COVID-19 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain ...In response to the ongoing COVID-19 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, i.e., "nanobodies." Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants at high potency, with IC values as low as 50 pM. Despite simultaneous binding to distinct epitopes, Beta and Omicron variants were more resistant to neutralization by the multimodular nanobodies, which highlights the importance of accounting for antigenic drift in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present an assay based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious diseases.
IMPORTANCE: Nanobodies, small protein binders derived from the camelid antibody, are highly potent inhibitors of respiratory viruses that offer several advantages over conventional antibodies as ...IMPORTANCE: Nanobodies, small protein binders derived from the camelid antibody, are highly potent inhibitors of respiratory viruses that offer several advantages over conventional antibodies as candidates for specific therapies, including high stability and low production costs. In this work, we leverage the unique properties of nanobodies and apply them as building blocks for new therapeutic and diagnostic tools. We report ultra-potent SARS-CoV-2 inhibition by engineered nanobodies comprising multiple modules in structure-guided combinations and develop nanobodies that carry signal molecules, allowing rapid detection of the SARS-CoV-2 spike protein. Our results highlight the potential of engineered nanobodies in the development of effective countermeasures, both therapeutic and diagnostic, to manage outbreaks of emerging viruses.
History
DepositionDec 7, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19068.png

Downloads & links

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Map

FileDownload / File: emd_19068.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap filtered to local resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 512 pix.
= 419.84 Å		0.82 Å/pix.
x 512 pix.
= 419.84 Å		0.82 Å/pix.
x 512 pix.
= 419.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.8612262 - 1.8432226
Average (Standard dev.)0.0013601353 (±0.027711302)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 419.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19068_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_19068_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_19068_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SARS-CoV-2 spike in complex with nanobody tri-TMH

EntireName: SARS-CoV-2 spike in complex with nanobody tri-TMH
Components
  • Complex: SARS-CoV-2 spike in complex with nanobody tri-TMH
    • Protein or peptide: SARS-CoV-2 Spike protein
    • Protein or peptide: Nanobody tri-TMH

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Supramolecule #1: SARS-CoV-2 spike in complex with nanobody tri-TMH

SupramoleculeName: SARS-CoV-2 spike in complex with nanobody tri-TMH / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Macromolecule #1: SARS-CoV-2 Spike protein

MacromoleculeName: SARS-CoV-2 Spike protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TGQCVNLTTR TQLPPAYTNS FTRGVYYPDK VFRSSVLHST QDLFLPFFSN VTWFHAIHVS GTNGTKRFDN PVLPFNDGVY FASTEKSNII RGWIFGTTLD SKTQSLLIVN NATNVVIKVC EFQFCNDPFL GVYYHKNNKS WMESEFRVYS SANNCTFEYV SQPFLMDLEG ...String:
TGQCVNLTTR TQLPPAYTNS FTRGVYYPDK VFRSSVLHST QDLFLPFFSN VTWFHAIHVS GTNGTKRFDN PVLPFNDGVY FASTEKSNII RGWIFGTTLD SKTQSLLIVN NATNVVIKVC EFQFCNDPFL GVYYHKNNKS WMESEFRVYS SANNCTFEYV SQPFLMDLEG KQGNFKNLRE FVFKNIDGYF KIYSKHTPIN LVRDLPQGFS ALEPLVDLPI GINITRFQTL LALHRSYLTP GDSSSGWTAG AAAYYVGYLQ PRTFLLKYNE NGTITDAVDC ALDPLSETKC TLKSFTVEKG IYQTSNFRVQ PTESIVRFPN ITNLCPFGEV FNATRFASVY AWNRKRISNC VADYSVLYNS ASFSTFKCYG VSPTKLNDLC FTNVYADSFV IRGDEVRQIA PGQTGKIADY NYKLPDDFTG CVIAWNSNNL DSKVGGNYNY LYRLFRKSNL KPFERDISTE IYQAGSTPCN GVEGFNCYFP LQSYGFQPTN GVGYQPYRVV VLSFELLHAP ATVCGPKKST NLVKNKCVNF NFNGLTGTGV LTESNKKFLP FQQFGRDIAD TTDAVRDPQT LEILDITPCS FGGVSVITPG TNTSNQVAVL YQDVNCTEVP VAIHADQLTP TWRVYSTGSN VFQTRAGCLI GAEHVNNSYE CDIPIGAGIC ASYQTQTNSP GSASSVASQS IIAYTMSLGA ENSVAYSNNS IAIPTNFTIS VTTEILPVSM TKTSVDCTMY ICGDSTECSN LLLQYGSFCT QLNRALTGIA VEQDKNTQEV FAQVKQIYKT PPIKDFGGFN FSQILPDPSK PSKRSFIEDL LFNKVTLADA GFIKQYGDCL GDIAARDLIC AQKFNGLTVL PPLLTDEMIA QYTSALLAGT ITSGWTFGAG AALQIPFAMQ MAYRFNGIGV TQNVLYENQK LIANQFNSAI GKIQDSLSST ASALGKLQDV VNQNAQALNT LVKQLSSNFG AISSVLNDIL SRLDPPEAEV QIDRLITGRL QSLQTYVTQQ LIRAAEIRAS ANLAATKMSE CVLGQSKRVD FCGKGYHLMS FPQSAPHGVV FLHVTYVPAQ EKNFTTAPAI CHDGKAHFPR EGVFVSNGTH WFVTQRNFYE PQIITTDNTF VSGNCDVVIG IVNNTVYDPL QPELDSFKEE LDKYFKNHTS PDVDLGDISG INASVVNIQK EIDRLNEVAK NLNESLIDLQ ELGKYEQGTG YIPEAPRDGQ AYVRKDGEWV LLSTFLGSGL EVLFQGPGSG RGVPHIVMVD AYKRYKGSGH HHHHHHH

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Macromolecule #2: Nanobody tri-TMH

MacromoleculeName: Nanobody tri-TMH / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDHPFTQVQL VETGGGLVQP GGSLRLSCAA SGFTFSSVYM NWVRQAPGKG PEWVSRISPN SGNIGYTDSV KGRFTISRDN AKNTLYLQMN NLKPEDTALY YCAIGLNLSS SSVRGQGTQV TVSSGGGGSG GGGSGGGGSG ...String:
MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDHPFTQVQL VETGGGLVQP GGSLRLSCAA SGFTFSSVYM NWVRQAPGKG PEWVSRISPN SGNIGYTDSV KGRFTISRDN AKNTLYLQMN NLKPEDTALY YCAIGLNLSS SSVRGQGTQV TVSSGGGGSG GGGSGGGGSG GGGSQVQLVE SGGGLVQAGG SLRLSCAASG FPVEVWRMEW YRQAPGKERE GVAAIESYGH GTRYADSVKG RFTISRDNAK NTVYLQMNSL KPEDTAVYYC NVYDDGQLAY HYDYWGQGTQ VTVSSGGGGS GGGGSGGGGS GGGGSQVQLV ESGGGLMQAG GSLRLSCAVS GRTFSTAAMG WFRQAPGKER EFVAAIRWSG GSAYYADSVK GRFTISRDKA KNTVYLQMNS LKYEDTAVYY CAQTHYVSYL LSDYATWPYD YWGQGTQVTV SS

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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: 8 / Details: 10 mM Tris pH 8, 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 274.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number real images: 3073 / Average exposure time: 5.0 sec. / Average electron dose: 1.38 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1)
Details: After refinement, the map was filtered to local resolution
Number images used: 26708
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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