X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation

Agdanowski, Matthew P.; Castells-Graells, Roger; Sawaya, Michael R.; Cascio, Duilio; Yeates, Todd O.; Arbing, Mark A.

doi:10.1107/S2053230X2400414X

Title: X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation

Journal Article · 19 May 2024 · Acta Crystallographica. Section F, Structural Biology Communications

DOI: https://doi.org/10.1107/S2053230X2400414X · OSTI ID:2352294

Agdanowski, Matthew P.;

;

; Cascio, Duilio; Yeates, Todd O.; Arbing, Mark A.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo' proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage–cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: NONE; FC02-02ER63421

OSTI ID:: 2352294

Alternate ID(s):: OSTI ID: 2472203

Journal Information:: Acta Crystallographica. Section F, Structural Biology Communications, Journal Name: Acta Crystallographica. Section F, Structural Biology Communications Journal Issue: 5 Vol. 80; ISSN ACSFEN; ISSN 2053-230X

Publisher:: International Union of Crystallography (IUCr)Copyright Statement

Country of Publication:: United Kingdom

Language:: English

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