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- EMDB-63071: SARS-CoV-2 spike glycoprotein trimer in prefusion form (1-RBD up ... -

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Basic information

Entry
Database: EMDB / ID: EMD-63071
TitleSARS-CoV-2 spike glycoprotein trimer in prefusion form (1-RBD up state)
Map data
Sample
  • Complex: SARS-CoV-2 spike glycoprotein
    • Protein or peptide: SARS-CoV-2 spike glycoprotein
Keywordsclass I membrane fusion glycoprotein / receptor-binding / viral entry / homotrimer / VIRAL PROTEIN
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsFukuhara H / Anraku Y / Kita S / Maenaka K
Funding support Japan, 6 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP20ae0101047 Japan
Japan Agency for Medical Research and Development (AMED)JP21fk0108463 Japan
Japan Agency for Medical Research and Development (AMED)JP22ama121037 Japan
Japan Agency for Medical Research and Development (AMED)JP223fa627005 Japan
Japan Science and TechnologyJPMJCR20H8 Japan
Japan Society for the Promotion of Science (JSPS)JP20H05873 Japan
CitationJournal: J Virol / Year: 2025
Title: ARNAX is an ideal adjuvant for COVID-19 vaccines to enhance antigen-specific CD4 and CD8 T-cell responses and neutralizing antibody induction.
Authors: Tomomi Kawakita / Toshiki Sekiya / Yayoi Kameda / Naoki Nomura / Marumi Ohno / Chimuka Handabile / Akari Yamaya / Hideo Fukuhara / Yuki Anraku / Shunsuke Kita / Shinsuke Toba / Hirotake ...Authors: Tomomi Kawakita / Toshiki Sekiya / Yayoi Kameda / Naoki Nomura / Marumi Ohno / Chimuka Handabile / Akari Yamaya / Hideo Fukuhara / Yuki Anraku / Shunsuke Kita / Shinsuke Toba / Hirotake Tsukamoto / Tomohiro Sawa / Hiroyuki Oshiumi / Yasushi Itoh / Katsumi Maenaka / Akihiko Sato / Hirofumi Sawa / Yasuhiko Suzuki / Lorena E Brown / David C Jackson / Hiroshi Kida / Misako Matsumoto / Tsukasa Seya / Masashi Shingai /
Abstract: ARNAX is a synthetic nucleotide-based Toll-like receptor 3 (TLR3) ligand that specifically stimulates the TLR3/TIR domain-containing adaptor molecule 1 (TICAM-1) pathway without activating ...ARNAX is a synthetic nucleotide-based Toll-like receptor 3 (TLR3) ligand that specifically stimulates the TLR3/TIR domain-containing adaptor molecule 1 (TICAM-1) pathway without activating inflammatory responses. ARNAX activates cellular immunity via cross-presentation; hence, its practical application has been demonstrated in cancer immunotherapy. Given the importance of cellular immunity in virus infections, ARNAX is expected to be a more effective vaccine adjuvant for virus infections than alum, an adjuvant approved for human use that mainly enhances humoral immunity. In the present study, the trimeric recombinant spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was prepared as a vaccine antigen and formulated with ARNAX. When T-cell and neutralizing antibody responses were evaluated in immunized mice, antigen formulated with ARNAX generated significantly larger numbers of antigenspecific CD4 and CD8 T cells, as well as higher titers of neutralizing antibodies, compared to antigen alone or antigen formulated with alum. In experiments where immunized mice were challenged with a SARS-CoV-2 mouse-adapted virus derived from the ancestral strain, immunization with antigen formulated with ARNAX reduced virus titers in the lungs at 3 days post-infection to a much greater extent than did immunization with either antigen alone or that formulated with alum. These results show that ARNAX potently enhances the levels of both cellular and humoral immunity above those seen with alum, providing significantly greater viral clearing responses. Thus, ARNAX may act as a useful adjuvant for prophylactic vaccines, particularly for viral infectious diseases.
IMPORTANCE: Cellular immunity is a critical immunological defense system against virus infections. However, aluminum salts, the most widely used adjuvant for vaccines for human use, do not promote ...IMPORTANCE: Cellular immunity is a critical immunological defense system against virus infections. However, aluminum salts, the most widely used adjuvant for vaccines for human use, do not promote strong cellular immunity. To prepare for the next pandemic of viral origin, the development of Th1-type adjuvants with low adverse reactions that induce cellular immunity is necessary. ARNAX is a TLR3 agonist consisting of DNA-RNA hybrid nucleic acid, which is expected to be an adjuvant that induces cellular immunity. The present study using a coronavirus disease 2019 mouse model demonstrated that ARNAX potently induces cellular immunity in addition to humoral immunity with minimal induction of inflammatory cytokines. Therefore, ARNAX has the potential to be used as a potent and welltolerated adjuvant for vaccines against pandemic viruses emerging in the future.
History
DepositionJan 10, 2025-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_63071.png

Downloads & links

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Map

FileDownload / File: emd_63071.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.89 Å/pix.
x 384 pix.
= 343.027 Å		0.89 Å/pix.
x 384 pix.
= 343.027 Å		0.89 Å/pix.
x 384 pix.
= 343.027 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8933 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.075
Minimum - Maximum-0.14518295 - 0.39233005
Average (Standard dev.)0.0010584301 (±0.012193581)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 343.0272 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_63071_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_63071_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_63071_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : SARS-CoV-2 spike glycoprotein

EntireName: SARS-CoV-2 spike glycoprotein
Components
  • Complex: SARS-CoV-2 spike glycoprotein
    • Protein or peptide: SARS-CoV-2 spike glycoprotein

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Supramolecule #1: SARS-CoV-2 spike glycoprotein

SupramoleculeName: SARS-CoV-2 spike glycoprotein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 420 KDa

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Macromolecule #1: SARS-CoV-2 spike glycoprotein

MacromoleculeName: SARS-CoV-2 spike glycoprotein / type: protein_or_peptide / ID: 1
Details: The transmembrane domain and cytoplasmic tail were replaced with a T4 trimerization motif, followed by the addition of an HRV 3C protease site, a Hexa-His tag, and a Strep-tag II at the C-terminus.
Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY ...String:
MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP GTNTSNQVAV LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PGSASSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQALN TLVKQLSSNF GAISSVLNDI LSRLDPPEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL QELGKYEQGS GYIPEAPRDG QAYVRKDGEW VLLSTFLGRS LEVLFQGPGH HHHHHHHSAW SHPQFEKGGG SGGGGSGGSA WSHPQFEK

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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

Concentration0.25 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
0.137 mol/LNaClsodium chloride
0.027 mol/LKClpotassium chloride
0.081 mol/LNa2HPO4disodium hydrogenphosphate
0.015 mol/LKH2PO4potassium dihydrogen phosphate

Details: PBS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: blotting time of 5 seconds and blotting force 5.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4792 pixel / Number grids imaged: 1 / Number real images: 3500 / Average exposure time: 0.9 sec. / Average electron dose: 53.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
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

Particle selectionNumber selected: 1448075
CTF correctionSoftware - Name: cryoSPARC (ver. 4.2.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio reconstruction in cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 31127
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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