5E0Q

Crystal structure of the Nup98 C-terminal domain bound to nanobody TP377

5E0Q の概要

• エントリーDOI: 10.2210/pdb5e0q/pdb
• 分子名称: Anti-Nup98 Nanobody TP377, Nuclear pore complex protein Nup98-Nup96 (3 entities in total)
• 機能のキーワード: nuclear pore complex, nuclear transport, autoproteolytic domain, antibody, transport protein
• 由来する生物種: Vicugna pacos; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 31169.71

構造登録者

Pleiner, T.,Trakhanov, S.,Goerlich, D. (登録日: 2015-09-29, 公開日: 2015-12-16, 最終更新日: 2024-01-10)

主引用文献

Pleiner, T.,Bates, M.,Trakhanov, S.,Lee, C.T.,Schliep, J.E.,Chug, H.,Bohning, M.,Stark, H.,Urlaub, H.,Gorlich, D.
Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.
Elife, 4:e11349-e11349, 2015

PubMed Abstract: Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.

PubMed: 26633879
DOI: 10.7554/eLife.11349

実験手法

X-RAY DIFFRACTION (1.9 Å)