Our results elucidate how fusion-loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. flaviviruses, can function to control contamination without appreciably recognizing mature virions. As these highly cross-reactive antibodies are often weakly neutralizing they also may contribute to antibody-dependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines. mosquito cells were produced in MEM supplemented with 10% fetal bovine serum (FBS) following standard cell culture procedures. Confluent cells were infected with DENV-2 at a multiplicity of contamination of 0.2 in the presence of 5% FBS. Medium was replaced 24 h after contamination with MEM made up of 20 mM NH4Cl. Cell culture supernatant was harvested 3 days after contamination and virus was purified as described for WNV. Complex formation, cryoEM and 3D image reconstructions Purified Ethisterone WNV particles were incubated with E53 Fab in the presence Rabbit Polyclonal to MRPL46 of 100 mM NaCl at 4C overnight, using a ratio of about five Fab fragments per E protein. Purified immature Ethisterone DENV particles were incubated with Fab at 37C for 30 min and then at 4C for 2 h, using a ratio of about two Fab fragments per E protein. Micrographs of the frozen-hydrated sample were recorded on Kodak (Rochester, NY) SO-163 films with a CM300 FEG transmission electron microscope (Philips, Eindhoven, The Netherlands). Images were taken at a nominal magnification of 47 000 and a total electron dose of 12C15 e?/?2. The cryoEM micrographs were digitized on a Nikon 9000 scanner (Tokyo, Japan) with a 6.35-m step size, and subsequently sets of four pixels were averaged to sample the specimen at 2.69 ? intervals. The program RobEM (Baker, 2004) was used to select a total of 4143 particles from 84 micrographs for the immature WNVCE53 Fab complex and a Ethisterone total of 2741 particles from 23 micrographs for the complex of immature DENV with E53 Fab. The defocus level was determined by fitting the theoretical microscope contrast transfer functions (CTFs) to the incoherent sum of the Fourier transforms of all particle images from each micrograph. The 3D reconstruction was computed using CTF phase-corrected images. The reconstruction was initiated by using a cryoEM density map of immature WNV as a model. The particle orientations were decided with SPIDER (Frank em et al /em , 1996), and the 3D electron density map was calculated with a modified version of XMIPP Ethisterone (Sorzano em et al /em , 2004) assuming icosahedral symmetry. Only 3927 and 2741 particles of the WNV and DENV complex, respectively, were selected to calculate the final 3D electron density maps. Selection was based on correlation with the model projections and stability of the particle centre position used. The resolution of the resultant map was estimated by comparing structure factors for the virus shell computed from two impartial half-data sets. The estimated resolution was based on determining the spacing frequency at which the correlation between the two impartial data sets became less than 0.5. One measure of the map quality is the resolution of the lipid leaflets. The above procedure did not give a good representation of the lipid bilayer in the immature WNVCE53 Fab reconstruction (Supplementary Physique 1). Thus, as an alternative reconstruction technique, the Polar Fourier Transform (PFT) (Baker and Cheng, 1996) reciprocal space procedure was used for both WNV and DENV. This gave considerably better representations of the membrane region of these viruses, but the quality of the density representing the glycoprotein was reduced. This might suggest that the PFT method is the better procedure indicating that the interpretation of the Fab density in the XMIPP reconstruction could be inaccurate. However, the excellent agreement of the cryoEM density of the E53 FabCvirus complex with the crystal structure of the soluble E ectodomain in complex with E53 Fab showed that the reconstruction based on the modified XMIPP procedure was accurate. Thus, the lack of a clear separation of the lipid bilayers in the membrane of the reconstructions is not the result of disorder in the lipid due to the E53 binding, but a limitation of the reconstruction techniques. Fitting of X-ray coordinates into the cryoEM electron density The fitting of the E and E53 Fab atomic coordinates to the immature virusFab complexes followed earlier procedures (Kaufmann em et al /em , 2006) using the program EMfit (Rossmann em et al /em , 2001). First, Ethisterone the E glycoprotein (PDB codes 3C6D (Li em et al /em , 2008) and 2OF6 (Zhang em et al /em , 2007) of DENV and WNV, respectively) and pr (PDB code 3C5X (Li em et al /em , 2008) for DENV and a model for the pr of WNV generated by SWISS-MODEL (Arnold em et al /em , 2006)) were fitted independently into the cryoEM density (Supplementary Table III). Each E molecule was.