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-Structure paper
タイトル | Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies). |
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ジャーナル・号・ページ | Proc Natl Acad Sci U S A, Vol. 118, Issue 41, Year 2021 |
掲載日 | 2021年10月12日 |
著者 | Xudong Wu / Tom A Rapoport / |
PubMed 要旨 | We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding ...We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis. |
リンク | Proc Natl Acad Sci U S A / PubMed:34620716 / PubMed Central |
手法 | EM (単粒子) / X線回折 |
解像度 | 1.83 - 3.6 Å |
構造データ | EMDB-24728, PDB-7rxc: EMDB-24729, PDB-7rxd: PDB-7r9d: |
化合物 | ChemComp-HOH: ChemComp-POV: |
由来 |
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キーワード | PROTEIN BINDING / scaffold / protein binder / STRUCTURAL PROTEIN / protein A / maltose-binding protein / Fab / nanobody / RBD |