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- PDB-9k75: SARS-CoV-2 related bat coronavirus BANAL-103 spike in the closed state -

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Entry
Database: PDB / ID: 9k75
TitleSARS-CoV-2 related bat coronavirus BANAL-103 spike in the closed state
ComponentsSpike glycoprotein
KeywordsVIRAL PROTEIN / SARS-CoV-2 related coronavirus / BANAL-103 / Spike
Biological speciesCoronaviridae (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsQingqing, L. / Xiao, C. / Xiaoning, L. / Yibing, Z. / Ru, L. / Zirui, K. / Didi, W. / Jiaxu, W. / Lili, L. / Junxia, Y. ...Qingqing, L. / Xiao, C. / Xiaoning, L. / Yibing, Z. / Ru, L. / Zirui, K. / Didi, W. / Jiaxu, W. / Lili, L. / Junxia, Y. / Jianxiang, S. / Shuiling, J. / Ying, P. / Na, Z. / Yushun, W. / Jian, S.
Funding support China, 2items
OrganizationGrant numberCountry
Other government32200113
Other government232301420013 China
CitationJournal: J Virol / Year: 2025
Title: Structural and functional constraints on spike activation and host protease utilization limit cell entry of SARS-CoV-2-related bat coronaviruses.
Authors: Qingqing Li / Xiao Cai / Xiaoning Li / Yibing Zhang / Ru Li / Zirui Kang / Didi Wan / Jiaxu Wang / Lili Li / Junxia Yang / Jianxiang Shi / Shuiling Jin / Xiangdong Sun / Ying Peng / Na Zang ...Authors: Qingqing Li / Xiao Cai / Xiaoning Li / Yibing Zhang / Ru Li / Zirui Kang / Didi Wan / Jiaxu Wang / Lili Li / Junxia Yang / Jianxiang Shi / Shuiling Jin / Xiangdong Sun / Ying Peng / Na Zang / Zhengkun Xie / Yushun Wan / Jian Shang /
Abstract: The persistent threat posed by SARS-CoV-2-related coronaviruses (SC2r-CoVs) in wildlife highlights the risk of zoonotic transmission. Cross-species infectivity is predominantly determined by spike (S) ...The persistent threat posed by SARS-CoV-2-related coronaviruses (SC2r-CoVs) in wildlife highlights the risk of zoonotic transmission. Cross-species infectivity is predominantly determined by spike (S) character and S-mediated cell entry. In this study, we systematically investigated BANAL-52 and BANAL-103, which exhibit the closest genetic proximity to SARS-CoV-2, focusing on their spike structures and functional characteristics. First, despite comparable receptor-binding domain (RBD)-ACE2 interactions, the spikes of BANAL-52 and BANAL-103 displayed significantly reduced ACE2 binding compared to SARS-CoV-2, suggesting impaired S activation. Second, Cryo-EM structural analyses revealed that BANAL-52 S is stabilized in a "locked" state through linoleic acid (LA) binding and an additional N370 glycan, whereas BANAL-103 S adopts a "closed" conformation due to a unique glycan network. Site-directed mutagenesis targeting the LA binding pocket confirmed that Y365 is related to S conformational transitions and viral entry. Third, both BANAL spikes relied predominantly on lysosomal proteases (e.g., cathepsins) for membrane fusion, unlike SARS-CoV-2, which utilizes a broader range of proteases (e.g., TMPRSS2 and furin). The introduction of a furin cleavage site enhanced the fusogenicity of BANAL spikes. Finally, sera from individuals who have recovered from SARS-CoV-2 effectively neutralized BANAL pseudoviruses, underscoring conserved antigenicity. Our findings elucidate structural and proteolytic barriers that restrict the zoonotic potential of these viruses and propose targeted surveillance strategies to preempt the emergence of SC2r-CoVs.
IMPORTANCE: The viral entry mechanisms, which are primarily related to the spike character, play a critical role in determining zoonotic potential. Among the currently identified SC2r-CoVs, BANAL-52 ...IMPORTANCE: The viral entry mechanisms, which are primarily related to the spike character, play a critical role in determining zoonotic potential. Among the currently identified SC2r-CoVs, BANAL-52 and BANAL-103 exhibit spike proteins with the highest sequence similarity to SARS-CoV-2, rendering them optimal models for comparative studies on S-mediated cell entry and cross-species transmission. In this study, we systematically investigated the molecular constraints governing the functionality of BANAL spikes, with a focus on S-ACE2 interactions, S activation, S structures, and host protease utilization. Notably, we resolved the cryo-EM structure of BANAL-52 S at neutral pH and the first cryo-EM structure of BANAL-103 S, revealing distinct glycan- and lipid-mediated stabilization of inactive states. Furthermore, cross-neutralization assays demonstrated that sera of convalescents from SARS-CoV-2 inhibited BANAL pseudovirus entry with an efficiency of approximately 80%, thereby highlighting conserved antigenic epitopes and informing the development of broad-spectrum therapeutic strategies against emerging SC2r-CoVs.
History
DepositionOct 23, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 16, 2025Provider: repository / Type: Initial release
Revision 1.0Jul 16, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 16, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.1Aug 6, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Sep 3, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)427,15763
Polymers413,8853
Non-polymers13,27260
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: Protein Spike glycoprotein


Mass: 137961.562 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Coronaviridae (virus) / Production host: Mammalia (mammals)
#2: 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.: 60 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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 interestY
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: BANAL-103 coronavirus spike / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Coronaviridae (virus)
Source (recombinant)Organism: Mammalia (mammals)
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: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52916 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 47.37 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002226931
ELECTRON MICROSCOPYf_angle_d0.485236654
ELECTRON MICROSCOPYf_chiral_restr0.04494335
ELECTRON MICROSCOPYf_plane_restr0.0034659
ELECTRON MICROSCOPYf_dihedral_angle_d4.65973897

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