7PHP

Structure of Multidrug and Toxin Compound Extrusion (MATE) transporter NorM by NabFab-fiducial assisted cryo-EM

7PHP の概要

• エントリーDOI: 10.2210/pdb7php/pdb
• EMDBエントリー: 13424
• 分子名称: Multidrug resistance protein NorM, NabFab HC, NabFab LC, ... (6 entities in total)
• 機能のキーワード: nabfab, anti-nanobody fab, fiducial, norm, mate, membrane protein
• 由来する生物種: Vibrio cholerae RC385; 詳細
• タンパク質・核酸の鎖数: 5
• 化学式量合計: 129152.98

構造登録者

Bloch, J.S.,Mukherjee, S.,Kowal, J.,Niederer, M.,Pardon, E.,Steyaert, J.,Kossiakoff, A.A.,Locher, K.P. (登録日: 2021-08-18, 公開日: 2021-09-01, 最終更新日: 2024-10-09)

主引用文献

Bloch, J.S.,Mukherjee, S.,Kowal, J.,Filippova, E.V.,Niederer, M.,Pardon, E.,Steyaert, J.,Kossiakoff, A.A.,Locher, K.P.
Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins.
Proc.Natl.Acad.Sci.USA, 118:-, 2021

PubMed Abstract: With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryogenic electron microscopy (cryo-EM) structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ∼50 kDa. Using an additional anti-Fab nanobody further facilitated reliable initial three-dimensional structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, is humanized, and can be conveniently expressed in in large amounts, it may be useful not only for structural biology but also for biomedical applications.

PubMed: 34782475
DOI: 10.1073/pnas.2115435118

実験手法

ELECTRON MICROSCOPY (3.47 Å)