Cryo-EM Structural Analysis of EBOV-Fab Complexes

Antibody Fab proteins were obtained by recombinant expression as described above or were generated by digestion of the corresponding IgG with papain (ThermoFisher). Fabs of EBOV-515 or EBOV-520 were added in 5 M excess to EBOV GP ΔTM and allowed to bind overnight at 4°C. Complexes were purified subsequently by size exclusion chromatography on an S200 Increase column (GE HealthCare), then deposited on copper mesh grids coated with carbon and stained 2% uranyl formate. Micrographs were collected using a 120KeV Tecnai Spirit with TVIPS TemCam F416 (4k x 4k) at a defocus of about 1.5e-06 defocus and a dose of 25e-/Å². Micrographs were collected using Leginon (Potter et al., 1999 (link)) and processed on Appion (Lander et al., 2009 (link)). Particles were picked using DoGpicker (Voss et al., 2009 (link)) and aligned with MSA/MRA (Ogura et al., 2003 (link)) where excess Fab or blurry particles were removed. An unbinned, clean dataset was deposited into Relion (Scheres, 2012 (link)) where 3D classification and refinement was performed. Figures were created in Chimera to compare EBOV complexes and show epitope location.

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Gilchuk P., Kuzmina N., Ilinykh P.A., Huang K., Gunn B.M., Bryan A., Davidson E., Doranz B.J., Turner H.L., Fusco M.L., Bramble M.S., Hoff N.A., Binshtein E., Kose N., Flyak A.I., Flinko R., Orlandi C., Carnahan R., Parrish E.H., Sevy A.M., Bombardi R.G., Singh P.K., Mukadi P., Muyembe-Tamfum J.J., Ohi M.D., Saphire E.O., Lewis G.K., Alter G., Ward A.B., Rimoin A.W., Bukreyev A, & Crowe JE J.r. (2018). Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein. Immunity, 49(2), 363-374.e10.

Publication 2018

papain S200 Increase column TVIPS TemCam F416

Antibody Carbon Chimera Copper Digestion Epitope Papain Proteins Size exclusion chromatography Uranyl formate

Corresponding Organization : Vanderbilt University

Other organizations : Ragon Institute of MGH, MIT and Harvard, Scripps Research Institute, University of California, Los Angeles, Children's National, University of Maryland, Baltimore, National Institute of Biomedical Research

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Variable analysis

independent variables

Addition of Fab proteins EBOV-515 or EBOV-520 to EBOV GP ΔTM in 5 M excess
Incubation of Fab-EBOV GP ΔTM complexes overnight at 4°C

dependent variables

Purification of Fab-EBOV GP ΔTM complexes by size exclusion chromatography
Deposition of complexes on copper mesh grids coated with carbon and stained with 2% uranyl formate
Imaging of the complexes using a 120KeV Tecnai Spirit with TVIPS TemCam F416 (4k x 4k) at a defocus of about 1.5e-06 and a dose of 25e-/Å^2
Particle picking, alignment, 3D classification, and refinement using computational methods (DoGpicker, MSA/MRA, Relion)

control variables

Recombinant expression or papain digestion of Fab proteins
Size of the S200 Increase column used for purification
Carbon coating and 2% uranyl formate staining of the copper mesh grids
Microscope settings (120KeV, 1.5e-06 defocus, 25e-/Å^2 dose)

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