SCIENCEADVANCES | RESEARCHARTICLE with a K3 direct detector (Gatan). A total of 10,235 movies were re- membranefilterusingaminiextruder(AvantiPolarLipids).Tore- corded with a calibrated pixel size of 0.826 Å, a defocus range of constitute proteoliposomes, the liposomes were destabilized by 2 −1to−2.5μm,and50frameswithatotaldoseof~60electrons/Å . 0.015% Triton X-100 and then incubated with purified Sialin at a ratio of 150:1 (wt/wt) for 20 min at room temperature. Bio-Beads Image processing, model building, and re昀椀nement SM-2 (150 mg; Bio-Rad Laboratories) were added to the mixture The data were processed with cryoSPARC (47). Patch motion cor- to absorb all detergents overnight at 4°C. The reconstituted proteo- rectionwasusedtocorrectthebeam-inducedmovement,andpatch liposomes were harvested by ultracentrifugation at 180,000g for 20 contrast transfer function was used to estimate contrast transfer minandthenresuspended in buffer D [20 mM MES (pH 5.6) and function parameters for each movie. A total of ~3 million particles 150mMNaCl].Proteoliposomesolution(100μl)foreachtransport 3 were automatically picked using “Blob Picker” and extracted with a was incubated with 10 nM [ H]Neu5Ac at room temperature. At box size of 320 × 320 pixels. After two rounds of reference-free 2D different time points, the proteoliposomes were filtered and classification, ab initio reconstruction, and heterogeneous refine- washed with ice-cold Buffer A. The filter membrane was scintilla- ment, ~800,000 particles were selected for further processing. 3D tion-counted. Each transport was repeated five times. The trans- classification without alignment was performed with the following ported amount was calculated with the assumption that the parameters: five classes, 6-Å target resolution, and principal com- orientation of reconstituted Sialin was 50:50. ponent analysis initiation mode. The final reconstruction was ob- tained with a particular class of 394,078 particles. After Immuno昀氀uorescence staining and microscopy imaging homogenesis refinement and nonuniform refinement, the map High Five cells with or without His-tagged Sialin expression were reached a resolution of ~3.7 Å. A soft mask around Sialin and fixed in 2% paraformaldehyde for 30 min at room temperature. half of the Fab was generated for a final round of local refinement After washing three times with PBS, cells were permeabilized with to produce the 3.4-Å-resolution map. The model was built in Coot 0.2% Triton X-100 for 30 min. Unpermeabilized or permeabilized (48). The AlphaFold-predicted model of Sialin was used as a guide cells were blocked in PBS containing 10% horseserumfor30minat together with several secondary structure prediction programs: roomtemperature.Cellswerethenincubatedwithprimaryantibod- Jpred (49) and PSSpred (50). The final model contains residues ies against 6×His (1:500; UBPBio) or anti-Sialin monoclonal anti- 32 to 68 and 102 to 488, missing flexible N-termini, C-termini, body 8B1 (1:2000) in 5% horse serum in PBS for 1 hour at room and the luminal loop between TM2 and TM3. The model was temperature. Cells were washed three times in PBS before costained refined in PHENIX (51). The quality of the model was assessed withsecondaryantibody(1:800;MolecularProbesAlexaFluor555) by MolProbity (52). Statistical details can be found in table S1. All andnucleidyeHoechst(1:5000;MolecularProbes)or4′,6-diamidi- superpositions of structures were calculated in the program UCSF no-2-phenylindole (Sigma-Aldrich; 1 μg/ml) for 1 hour at room ChimerawiththealignmentalgorithmofNeedleman-Wunschand temperature.StainedcellswerewashedthreetimesinPBS,followed BLOSUM-62matrix (53). by microscopy imaging with an Olympus FV1000 FCS/RICS con- focal microscope or a Keyence BZ-X710 All-in-One Fluorescence Cellular transport assay Microscope. Sialic acid uptake was measured in High-Five cells expressing wild- type and various mutations of Sialin. Specifically, 200 μl of High- Five cells (0.4 × 106 count/ml) was seeded in 24-well plates in Supplementary Materials Grace’s Insect Medium supplemented with fetal bovine serum This PDF 昀椀le includes: and infected with the corresponding baculoviruses for 24 hours. Figs. S1 to S13 Cells were washed twice with buffer C [20 mM MES (pH 5.6), 5 Tables S1 and S2 mMglucose,150mMNaCl,and1mMMgSO ]andthenincubated References 3 4 with[ H]Neu5Ac(0.05μCi,assumedtobe10nMin250μlofbuffer View/request a protocol for this paper from Bio-protocol. C) for 15 min at room temperature. After washing twice with ice- cold buffer at pH 7.4, the radioactivity in the cells was counted by liquid scintillation counting using a PerkinElmer Tri-Carb 2910 TR REFERENCESANDNOTES machine. Each transport was measured five times independently. 1. D. Adams, M. Wasserstein, in GeneReviews((R)), M. P. Adam, H. H. Ardinger, R. A. Pagson, The expression level of wild-type and mutant Sialin in High-Five S. E. Wallace, L. J. H. Bean, K. W. Gripp, G. M. Mirzaa, A. Amemiya, Eds. (University of cells was determined by Western blot analysis, which was used to Washington, Seattle, 1993). normalize the substrate transport measurements. The transported 2. T. Angata, A. 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