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- PDB-9z1m: Cryo-EM structure of KP.3 spike in complex with Nb9B -

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Basic information

Entry
Database: PDB / ID: 9z1m
TitleCryo-EM structure of KP.3 spike in complex with Nb9B
Components
  • KP.3 spike
  • Nanosota-9B
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / SARS-CoV-2 / entry / VIRAL PROTEIN-IMMUNE SYSTEM complex
Biological speciesVicugna pacos (alpaca)
Severe acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsYe, G. / Bu, F. / Liu, B. / Li, F.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI171954 United States
CitationJournal: PLoS Pathog / Year: 2026
Title: Update and reuse: Structure-guided nanobody evolution against SARS-CoV-2 escape.
Authors: Fan Bu / Divyasha Saxena / Hailey Turner-Hubbard / Amy Delaney / Lalit Batra / Charlie Fricke / Skyler Moye / Abhishek Verma / Stanley Perlman / Janarjan Bhandari / Bin Liu / Gang Ye / Jian Zheng / Fang Li /
Abstract: SARS-CoV-2 continues to accumulate spike mutations that erode the efficacy of antibody therapeutics. The Q493E mutation in the spike RBD, present in recent Omicron subvariants, enables escape from ...SARS-CoV-2 continues to accumulate spike mutations that erode the efficacy of antibody therapeutics. The Q493E mutation in the spike RBD, present in recent Omicron subvariants, enables escape from many antibodies and nanobodies, including our Nanosota-9A nanobody, which neutralizes Omicron JN.1 (Q493) but not KP.3 (E493). To address this, we applied a structure-guided in vitro evolution strategy to engineer Nanosota-9A, generating Nanosota-9B, which binds the KP.3 RBD with high affinity but shows reduced binding to JN.1 RBD. To regain breadth, we engineered a bispecific nanobody combining Nanosota-9A and -9B, which effectively neutralizes both JN.1 and KP.3 in infection assays. Our results provide proof of concept for an "update and reuse" strategy: applying structure-guided engineering to update and reuse validated nanobodies to overcome variant escape. This strategy offers a practical path to maintain therapeutic coverage as the virus evolves, supporting more efficient use of research resources and faster responses to emerging variants.
History
DepositionNov 4, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 13, 2026Provider: repository / Type: Initial release
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Revision 1.1Jun 24, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Nanosota-9B
E: Nanosota-9B
F: Nanosota-9B
B: KP.3 spike
C: KP.3 spike
A: KP.3 spike
hetero molecules


Theoretical massNumber of molelcules
Total (without water)469,40221
Polymers463,8496
Non-polymers5,55315
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody Nanosota-9B


Mass: 16304.608 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)
#2: Protein KP.3 spike


Mass: 138311.781 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Homo sapiens (human)
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: KP.3 spike in complex with Nb9B / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 750 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
9PHENIX1.21.1_5286model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95868 / Symmetry type: POINT
Atomic model buildingPDB-ID: 9CO8

9co8


Accession code: 9CO8 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.06 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00529443
ELECTRON MICROSCOPYf_angle_d0.91240049
ELECTRON MICROSCOPYf_dihedral_angle_d13.36210563
ELECTRON MICROSCOPYf_chiral_restr0.0594540
ELECTRON MICROSCOPYf_plane_restr0.0075159

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