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Structure paper

TitleProduction and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct.
Journal, issue, pagesJ Struct Biol X, Vol. 11, Page 100123, Year 2025
Publish dateFeb 11, 2025
AuthorsSuruchi Singh / Yi Liu / Meghan Burke / Vamseedhar Rayaprolu / Stephen E Stein / S Saif Hasan /
PubMed AbstractThe SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the ...The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on -glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination.
External linksJ Struct Biol X / PubMed:40046771 / PubMed Central
MethodsEM (single particle)
Resolution2.32 - 2.99 Å
Structure data

44062

9b0y

EMDB-44062, PDB-9b0y:
SARS CoV-2 Spike protein Ectodomain with internal tag, all RBD-down conformation
Method: EM (single particle) / Resolution: 2.32 Å

44116

9b2v

EMDB-44116, PDB-9b2v:
SARS CoV-2 Spike protein Ectodomain with internal tag, 1RBD-up conformation
Method: EM (single particle) / Resolution: 2.55 Å

45893

9css

EMDB-45893, PDB-9css:
Cryo-EM structure of SARS-CoV-2 spike protein Ecto-domain with internal tag, 1UP RBD conformation
Method: EM (single particle) / Resolution: 2.72 Å

45895

9ct2

EMDB-45895, PDB-9ct2:
Cryo-EM structure of SARS-CoV-2 spike protein Ecto-domain with internal tag, All RBD down conformation, State-3
Method: EM (single particle) / Resolution: 2.7 Å

45965

9cvh

EMDB-45965, PDB-9cvh:
Cryo-EM structure of SARS-CoV-2 spike protein Ecto-domain with internal tag, 1RBD UP, State-2
Method: EM (single particle) / Resolution: 2.76 Å

45987

9cxe

EMDB-45987, PDB-9cxe:
SARS CoV-2 Spike protein Ectodomain with internal tag, all RBD-down conformation -C1
Method: EM (single particle) / Resolution: 2.48 Å

48839

9n2l

EMDB-48839, PDB-9n2l:
Cryo-EM structure of locally refined up conformation of SARS-CoV-2 spike protein Receptor Binding Domain
Method: EM (single particle) / Resolution: 2.99 Å

Chemicals

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

Source
  • severe acute respiratory syndrome coronavirus 2
  • severe acute respiratory syndrome coronavirus
KeywordsVIRAL PROTEIN / SARS CoV-2 Spike protein Ectodomain with internal tag / SARS-CoV-2 Spike protein Ectodomain with internal tag / SARS-CoV-2 Spike protein / Receptor Binding Domain

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