Dataset Information

Single particle cryo-EM dataset of extended cowpea chlorotic mottle virus at pH 7.6 in plunge-frozen vitrified ice

ABSTRACT:

SUBMITTER: Sarah Victoria Barrass

PROVIDER: EMPIAR-11489 | biostudies-other |

REPOSITORIES: biostudies-other

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Publications

Fast viral dynamics revealed by microsecond time-resolved cryo-EM.

Harder Oliver F OF Barrass Sarah V SV Drabbels Marcel M Lorenz Ulrich J UJ

Nature communications 20230913 1

Observing proteins as they perform their tasks has largely remained elusive, which has left our understanding of protein function fundamentally incomplete. To enable such observations, we have recently proposed a technique that improves the time resolution of cryo-electron microscopy (cryo-EM) to microseconds. Here, we demonstrate that microsecond time-resolved cryo-EM enables observations of fast protein dynamics. We use our approach to elucidate the mechanics of the capsid of cowpea chlorotic ...[more]

PMID: 37704664

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Project description:Background & methodsWithin the last decade Virus-Like Particles (VLPs) have increasingly received attention from scientists for their use as a carrier of (peptide) molecules or as scaffold to present epitopes for use in subunit vaccines. To test the feasibility of Cowpea chlorotic mottle virus (CCMV) particles as a scaffold for epitope presentation and identify sites for epitope fusion or insertion that would not interfere with virus-like-particle formation, chimeric CCMV coat protein (CP) gene constructs were engineered, followed by expression in E. coli and assessment of VLP formation. Various constructs were made encoding a 6x-His-tag, or selected epitopes from Influenza A virus [IAV] (M2e, HA) or Foot and Mouth Disease Virus [FMDV] (VP1 and 2C). The epitopes were either inserted 1) in predicted exposed loop structures of the CCMV CP protein, 2) fused to the amino- (N) or carboxyl-terminal (C) ends, or 3) to a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.ResultsHigh levels of insoluble protein expression, relative to proteins from the entire cell lysate, were obtained for CCMV CP and all chimeric derivatives. A straightforward protocol was used that, without the use of purification columns, successfully enabled CCMV CP protein solubilization, reassembly and subsequent collection of CCMV CP VLPs. While insertions of His-tag or M2e (7-23 aa) into the predicted external loop structures did abolish VLP formation, high yields of VLPs were obtained with all fusions of His-tag or various epitopes (13- 27 aa) from IAV and FMDV at the N- or C-terminal ends of CCMV CP or N∆24-CP. VLPs derived from CCMV CP still encapsulated RNA, while those from CCMV CP-chimera containing a negatively charged N-terminal domain had lost this ability. The usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines was demonstrated with the synthesis of chimeric CCMV VLPs containing selected sequences from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus at both termini of the CP protein.ConclusionsCCMV VLPs can be successfully exploited as scaffold for epitope fusions up to 31 aa at the N- and C-terminus, and at a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.

Dataset Information

Single particle cryo-EM dataset of extended cowpea chlorotic mottle virus at pH 7.6 in plunge-frozen vitrified ice

Publications

Fast viral dynamics revealed by microsecond time-resolved cryo-EM.

Similar Datasets

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