Protein samples were purified into crystallization buffer [10 mM tris (pH 8.0) and 30 mM NaCl]. The final protein concentration was approximately 10 mg/ml. Commercial crystallization screen solutions were dispensed into 96-well plates using an Art-Robbins Instruments Griffon dispensing robot (Alpha Biotech Ltd.) in sitting drop vapor diffusion format. Drops containing 200 nl of screen solution and 200 nl of protein solution were equilibrated against a reservoir of 60 μl of crystallization solution. The plates were sealed and incubated at 18°C.

Crystals of HAdV-D26K appeared in PACT Premier conditions B01 and B04 [0.1 M MIB (malonic acid, imidazole, and boric acid) and 25% w/v polyethylene glycol 1500, pH 4.0 and pH 8.0, respectively], within 1 to 7 days. Crystals were then soaked in reservoir solution containing Neu5Ac (Sigma-Aldrich, cat. no. A2388) at a final concentration of 10 mM and incubated overnight before harvest. Crystals were cryoprotected with reservoir solution to which ethylene glycol was added at a final concentration of 25%. Crystals were harvested in thin plastic loops and stored in liquid nitrogen for transfer to the synchrotron. Data were collected at Diamond Light Source beamline I04, running at a wavelength of 0.9795 Å. During data collection, crystals were maintained in a cold air stream at 100°K. Dectris PILATUS 6M detectors recorded the diffraction patterns, which were analyzed and reduced with XDS (55), Xia2, DIALS (56), and autoProc (57). Scaling and merging data were completed with Pointless, Aimless and Truncate from the CCP4 package (58). Structures were solved with Phaser, COOT was used to correct the sequences and adjust the models, and REFMAC5 was used to refine the structures and calculate maps. Graphical representations were prepared with PyMOL. Reflection data and final models were deposited in the PDB database with accession codes 6QU6, 6QU8, and 6FJO. Full crystallographic refinement statistics are given in table S1.

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