KDEL sequence binding / COPI-dependent Golgi-to-ER retrograde traffic / COPI-coated vesicle membrane / ER retention sequence binding / protein retention in ER lumen / COPI-mediated anterograde transport / maintenance of protein localization in endoplasmic reticulum / cis-Golgi network / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / IgG binding ...KDEL sequence binding / COPI-dependent Golgi-to-ER retrograde traffic / COPI-coated vesicle membrane / ER retention sequence binding / protein retention in ER lumen / COPI-mediated anterograde transport / maintenance of protein localization in endoplasmic reticulum / cis-Golgi network / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / IgG binding / carbohydrate transmembrane transporter activity / endoplasmic reticulum to Golgi vesicle-mediated transport / protein transport / periplasmic space / Golgi membrane / endoplasmic reticulum membrane / endoplasmic reticulum / extracellular region / membrane 類似検索 - 分子機能
ER lumen protein retaining receptor / ER lumen protein retaining receptor / ER lumen protein retaining receptor signature 1. / ER lumen protein retaining receptor signature 2. / Octapeptide repeat / Octapeptide repeat / Protein A, Ig-binding domain / B domain / IgG-binding B / B domain ...ER lumen protein retaining receptor / ER lumen protein retaining receptor / ER lumen protein retaining receptor signature 1. / ER lumen protein retaining receptor signature 2. / Octapeptide repeat / Octapeptide repeat / Protein A, Ig-binding domain / B domain / IgG-binding B / B domain / M protein-type anchor domain / GA-like domain / GA-like domain / Lysin motif / LysM domain superfamily / LysM domain / LysM domain profile. / LysM domain / Immunoglobulin/albumin-binding domain superfamily / YSIRK type signal peptide / YSIRK Gram-positive signal peptide / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / LPXTG cell wall anchor domain / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein 類似検索 - ドメイン・相同性
Maltodextrin-binding protein / Immunoglobulin G-binding protein G / Immunoglobulin G-binding protein A / ER lumen protein-retaining receptor 2 類似検索 - 構成要素
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM052586
米国
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2021 タイトル: Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies). 著者: Xudong Wu / Tom A Rapoport / 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.