+
検索条件

キーワード
著者・登録者
ジャーナル
IF	>30 >20 >10 >5 all

-
Structure paper

タイトル	Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins.
ジャーナル・号・ページ	Proc Natl Acad Sci U S A, Vol. 118, Issue 47, Year 2021
掲載日	2021年11月23日
著者	Joël S Bloch / Somnath Mukherjee / Julia Kowal / Ekaterina V Filippova / Martina Niederer / Els Pardon / Jan Steyaert / Anthony A Kossiakoff / Kaspar P Locher /
PubMed 要旨	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 ...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.
リンク	Proc Natl Acad Sci U S A / PubMed:34782475 / PubMed Central
手法	EM (単粒子) / X線回折
解像度	3.19 - 8.45 Å
構造データ	13424 7php EMDB-13424, PDB-7php: Structure of Multidrug and Toxin Compound Extrusion (MATE) transporter NorM by NabFab-fiducial assisted cryo-EM 手法: EM (単粒子) / 解像度: 3.47 Å EMDB-13425: Map of complex of Vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody with fulcrum in center of micelle 手法: EM (単粒子) / 解像度: 3.68 Å 13426 7phq EMDB-13426, PDB-7phq: Structure of homo-dimeric Staphylococcus capitis divalent metal ion transporter (DMT) by NabFab-fiducial assisted cryo-EM 手法: EM (単粒子) / 解像度: 8.45 Å 13438 7pij EMDB-13438, PDB-7pij: Structure of Staphylococcus capitis divalent metal ion transporter (DMT) by NabFab-fiducial assisted cryo-EM 手法: EM (単粒子) / 解像度: 3.78 Å PDB-7rth: Crystal structure of an anti-lysozyme nanobody in complex with an anti-nanobody Fab "NabFab" 手法: X-RAY DIFFRACTION / 解像度: 3.19 Å
化合物	ChemComp-HOH: WATER ChemComp-PO4: PHOSPHATE ION / ホスファ-ト ChemComp-CL: Unknown entry / クロリド ChemComp-GOL: GLYCEROL / グリセロ-ル ChemComp-PEG: DI(HYDROXYETHYL)ETHER / ジエチレングリコ-ル
由来	vibrio cholerae rc385 (コレラ菌) synthetic construct (人工物) lama glama (ラマ) staphylococcus capitis (バクテリア) homo sapiens (ヒト)
キーワード	MEMBRANE PROTEIN / NabFab / anti-nanobody Fab / fiducial / NorM / MATE / DMT / IMMUNE SYSTEM / Antigen / FAB / Single-Domain Antibody