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- EMDB-11954: SARS-CoV-2 S 2P trimer in complex with monovalent DARPin R2 (State 2) -

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Basic information

Entry
Database: EMDB / ID: EMD-11954
TitleSARS-CoV-2 S 2P trimer in complex with monovalent DARPin R2 (State 2)
Map dataStructure of the SARS-CoV-2 S 2P trimer in complex with mono-DARPin #3 (State 2)
Sample
  • Complex: SARS-CoV-2 S 2P trimer in complex with monovalent DARPin R2 (State 2)
    • Complex: SARS-CoV-2 spike glycoprotein
      • Protein or peptide: SARS-CoV-2 spike glycoprotein
    • Complex: monovalent DARPin R2
      • Protein or peptide: monovalent DARPin R2
Biological speciesSevere acute respiratory syndrome-related coronavirus / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.6 Å
AuthorsHurdiss DL / Drulyte I
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Other private Switzerland
CitationJournal: Nat Biotechnol / Year: 2022
Title: The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants.
Authors: Sylvia Rothenberger / Daniel L Hurdiss / Marcel Walser / Francesca Malvezzi / Jennifer Mayor / Sarah Ryter / Hector Moreno / Nicole Liechti / Andreas Bosshart / Chloé Iss / Valérie Calabro ...Authors: Sylvia Rothenberger / Daniel L Hurdiss / Marcel Walser / Francesca Malvezzi / Jennifer Mayor / Sarah Ryter / Hector Moreno / Nicole Liechti / Andreas Bosshart / Chloé Iss / Valérie Calabro / Andreas Cornelius / Tanja Hospodarsch / Alexandra Neculcea / Thamar Looser / Anja Schlegel / Simon Fontaine / Denis Villemagne / Maria Paladino / Dieter Schiegg / Susanne Mangold / Christian Reichen / Filip Radom / Yvonne Kaufmann / Doris Schaible / Iris Schlegel / Christof Zitt / Gabriel Sigrist / Marcel Straumann / Julia Wolter / Marco Comby / Feyza Sacarcelik / Ieva Drulyte / Heyrhyoung Lyoo / Chunyan Wang / Wentao Li / Wenjuan Du / H Kaspar Binz / Rachel Herrup / Sabrina Lusvarghi / Sabari Nath Neerukonda / Russell Vassell / Wei Wang / Julia M Adler / Kathrin Eschke / Mariana Nascimento / Azza Abdelgawad / Achim D Gruber / Judith Bushe / Olivia Kershaw / Charles G Knutson / Kamal K Balavenkatraman / Krishnan Ramanathan / Emanuel Wyler / Luiz Gustavo Teixeira Alves / Seth Lewis / Randall Watson / Micha A Haeuptle / Alexander Zürcher / Keith M Dawson / Daniel Steiner / Carol D Weiss / Patrick Amstutz / Frank J M van Kuppeveld / Michael T Stumpp / Berend-Jan Bosch / Olivier Engler / Jakob Trimpert /
Abstract: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad ...The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).
History
DepositionNov 18, 2020-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateAug 17, 2022-
Current statusAug 17, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_11954.png

Downloads & links

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Map

FileDownload / File: emd_11954.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the SARS-CoV-2 S 2P trimer in complex with mono-DARPin #3 (State 2)
Voxel sizeX=Y=Z: 1.045 Å
Density
Contour LevelBy AUTHOR: 0.00687
Minimum - Maximum-0.011359388 - 0.032948248
Average (Standard dev.)0.00014977073 (±0.0015069065)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 417.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map.

Fileemd_11954_additional_1.map
AnnotationUnsharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1.

Fileemd_11954_half_map_1.map
AnnotationHalf map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2.

Fileemd_11954_half_map_2.map
AnnotationHalf map 2.
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 S 2P trimer in complex with monovalent DARPin R2 (State 2)

EntireName: SARS-CoV-2 S 2P trimer in complex with monovalent DARPin R2 (State 2)
Components
  • Complex: SARS-CoV-2 S 2P trimer in complex with monovalent DARPin R2 (State 2)
    • Complex: SARS-CoV-2 spike glycoprotein
      • Protein or peptide: SARS-CoV-2 spike glycoprotein
    • Complex: monovalent DARPin R2
      • Protein or peptide: monovalent DARPin R2

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Supramolecule #1: SARS-CoV-2 S 2P trimer in complex with monovalent DARPin R2 (State 2)

SupramoleculeName: SARS-CoV-2 S 2P trimer in complex with monovalent DARPin R2 (State 2)
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 464 KDa

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Supramolecule #2: SARS-CoV-2 spike glycoprotein

SupramoleculeName: SARS-CoV-2 spike glycoprotein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome-related coronavirus
Strain: Severe acute respiratory syndrome coronavirus 2 (Wuhan-Hu-1)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK-293T

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Supramolecule #3: monovalent DARPin R2

SupramoleculeName: monovalent DARPin R2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: SARS-CoV-2 spike glycoprotein

MacromoleculeName: SARS-CoV-2 spike glycoprotein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome-related coronavirus
Strain: Severe acute respiratory syndrome coronavirus 2 (Wuhan-Hu-1)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MFVFLVLLPL VSSQCVNLTT RTQLPPAYT NSFTRGVYYP D KVFRSSVL HSTQDLFLPF FS NVTWFHA IHVSGTNGTK RFD NPVLPF NDGVYFASTE KSNI IRGWI FGTTLDSKTQ SLLIV NNAT NVVIKVCEFQ FCNDPF LGV YYHKNNKSWM ESEFRVY SS ...String:
MFVFLVLLPL VSSQCVNLTT RTQLPPAYT NSFTRGVYYP D KVFRSSVL HSTQDLFLPF FS NVTWFHA IHVSGTNGTK RFD NPVLPF NDGVYFASTE KSNI IRGWI FGTTLDSKTQ SLLIV NNAT NVVIKVCEFQ FCNDPF LGV YYHKNNKSWM ESEFRVY SS ANNCTFEYVS QPFLMDLE G KQGNFKNLRE FVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDL PIGINITRFQ T LLALHRSY LTPGDSSSGW TA GAAAYYV GYLQPRTFLL KYN ENGTIT DAVDCALDPL SETK CTLKS FTVEKGIYQT SNFRV QPTE SIVRFPNITN LCPFGE VFN ATRFASVYAW NRKRISN CV ADYSVLYNSA SFSTFKCY G VSPTKLNDLC FTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDF TGCVIAWNSN N LDSKVGGN YNYLYRLFRK SN LKPFERD ISTEIYQAGS TPC NGVEGF NCYFPLQSYG FQPT NGVGY QPYRVVVLSF ELLHA PATV CGPKKSTNLV KNKCVN FNF NGLTGTGVLT ESNKKFL PF QQFGRDIADT TDAVRDPQ T LEILDITPCS FGGVSVITP GTNTSNQVAV LYQDVNCTEV PVAIHADQL TPTWRVYSTG S NVFQTRAG CLIGAEHVNN SY ECDIPIG AGICASYQTQ TNS PAAARS VASQSIIAYT MSLG AENSV AYSNNSIAIP TNFTI SVTT EILPVSMTKT SVDCTM YIC GDSTECSNLL LQYGSFC TQ LNRALTGIAV EQDKNTQE V FAQVKQIYKT PPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLA DAGFIKQYGD C LGDIAARD LICAQKFNGL TV LPPLLTD EMIAQYTSAL LAG TITSGW TFGAGAALQI PFAM QMAYR FNGIGVTQNV LYENQ KLIA NQFNSAIGKI QDSLSS TAS ALGKLQDVVN QNAQALN TL VKQLSSNFGA ISSVLNDI L SRLDPPEAEV QIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKM SECVLGQSKR V DFCGKGYH LMSFPQSAPH GV VFLHVTY VPAQEKNFTT APA ICHDGK AHFPREGVFV SNGT HWFVT QRNFYEPQII TTDNT FVSG NCDVVIGIVN NTVYDP LQP ELDSFKEELD KYFKNHT SP DVDLGDISGI NASVVNIQ K EIDRLNEVAK NLNESLIDL LIKGSGYIPE APRDGQAYVR KDGEWVLLS TFLIKLVPRG S LEWSHPQF EK

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Macromolecule #2: monovalent DARPin R2

MacromoleculeName: monovalent DARPin R2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MRGSHHHHHH GSDLGKKLLQ AARAGQLDEV RELLKAGADV NAKDREGKTP LHVAAQEGHL EIVEVLLKAG ADVNAKDVWG RTPLHLAAWI GHLEIVEVLL KAGADVNAKD VSGATPLHAA ALHGHLEIVE VLLNAGADVN AQDKSGKTPA DLAARAGHQD IAEVLQKAA

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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

Concentration2.5 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsData collected with a 30 degree stage tilt.
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 3024 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 123833
CTF correctionSoftware - Name: RELION (ver. 3.1)
Startup modelType of model: EMDB MAP
EMDB ID:

21457

Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: CTFFIND (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 21612
FSC plot (resolution estimation)

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