6B14

Crystal structure of Spinach RNA aptamer in complex with Fab BL3-6S97N

Summary for 6B14

• Entry DOI: 10.2210/pdb6b14/pdb
• Descriptor: Heavy chain of Fab BL3-6S97N, Light chain of Fab BL3-6S97N, RNA (86-MER), ... (5 entities in total)
• Functional Keywords: spinach rna aptamer, antibody engineering, chaperone-assisted rna crystallography, immune system-rna complex, immune system/rna
• Biological source: Mus musculus; More
• Total number of polymer chains: 3
• Total formula weight: 74239.93

Authors

DasGupta, S.,Shelke, S.A.,Piccirilli, J.A. (deposition date: 2017-09-16, release date: 2017-12-27, Last modification date: 2024-10-09)

Primary citation

Koirala, D.,Shelke, S.A.,Dupont, M.,Ruiz, S.,DasGupta, S.,Bailey, L.J.,Benner, S.A.,Piccirilli, J.A.
Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography.
Nucleic Acids Res., 46:2624-2635, 2018

PubMed Abstract: Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5'-GNGACCC-3' consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab-RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography.

PubMed: 29309709
DOI: 10.1093/nar/gkx1292

Experimental method

X-RAY DIFFRACTION (1.64 Å)