5E0Q

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

Summary for 5E0Q

• Entry DOI: 10.2210/pdb5e0q/pdb
• Descriptor: Anti-Nup98 Nanobody TP377, Nuclear pore complex protein Nup98-Nup96 (3 entities in total)
• Functional Keywords: nuclear pore complex, nuclear transport, autoproteolytic domain, antibody, transport protein
• Biological source: Vicugna pacos; More
• Total number of polymer chains: 2
• Total formula weight: 31169.71

Authors

Pleiner, T.,Trakhanov, S.,Goerlich, D. (deposition date: 2015-09-29, release date: 2015-12-16, Last modification date: 2024-01-10)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.9 Å)