Supplementary Materials and Methods

Electron-microscopy analysis

MG1655 E. coli wild-type and zapE strains were observed by EM for immunodetection of ZapE. For immuno-EM, bacteria were fixed with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4), and embedded in 12% gelatin. Blocks were infiltrated with 2.3 M sucrose for cryoprotection, mounted on specimen holders and frozen in liquid nitrogen. Cryosections were performed with a Leica EM UC6/FC6 Microtome (Leica Microsystems, Vienna, Austria). A labeling was performed on thawed cryosections using antibody directed against ZapE, which is recognized by protein A gold. Cryosections were labeled first with an -ZapE rabbit polyclonal antibody at 1/1000 dilution, then with protein-A gold-10 nm diluted at 1/60 obtained from Utrecht University (Utrecht, The Netherlands) (1, 2).The grids were viewed on a Jeol JEM 1010 (Japan) transmission electron microscope at 80 kV and Images were taken using a KeenView camera (Soft Imaging System, Lakewood, CO, USA) using iTEM5.0 software (Soft Imaging System GmbH).

SAXS structural analysis

Small Angle X-ray Scattering Experiments. Two different data sets were collected at 10°C for various ZapE concentrations ranging from 0.32 to 5.35 mg/ml at the BM29 BioSAXS beamline at the ESRF synchrotron(3). Samples were loaded to 1.8 mm diameter quartz capillary with a few tens of micron wall thickness using continuous flow during data collection. The SAXS data were recorded using a Pilatus 1 M detector at a sample to detector distance of 2.43 m, covering the range of momentum transfer 0.05<s<0.5Å−1 (s = 4πsin(θ)/λ where 2θ is the scattering angle and λ = 0.931. is the X-ray wavelength). To evaluate radiation damage, ten successive 10 sec exposures of each sample were recorded. The PRIMUS software was used for normalization of the intensity to the incident beam, correction of the detector response, evaluation of the radiation damage and sample monodispersity, buffer subtraction, extrapolation to zero concentration, data scaling and merging. Estimation of the radius of gyration (Rg) and of the forward scattering intensity I(0) (proportional to the number of electrons in the particle) took place also in PRIMUS using the Guinier approximation (Rice, 1956). MW calibration was based on scattering data from a 5 mg/ml BSA solution. The indirect Fourier transform package GNOM was used for the evaluation of the maximum particle diameter (Dmax) and the calculation of the pair distribution [P(r)] function.

Ab initio shape determination. Simulated annealing methods (4)based on the small-angle X-ray scattering data were used to produced several three-dimensional models of dammy atoms (DAMMIN) or dammy residues [GASBOR, (5)]. No symmetry restrictions were applied as ZapE was found monomeric in solution. Initial particle shape was set to unknown. For GASBOR runs 348 dummy residues were used corresponding to the number of amino acids present in ZapE polypeptide. The scattering profiles were used up to smax = 0.24 .−1. The low-resolution models obtained from different runs were compared using the program DAMAVER to give an estimate of the reproducibility of the results inferred.

Integrative structural modeling. Initial rigid structural domains were generated by the fold recognition server PHYRE (6)based on the ZapE sequence. The higher scoring templates were used to produce rigid domains to be imported on CORAL [MS in preparation, CORAL combines the algorithms of SASREF and BUNCH (7)]. The relevant position of the rigid domains was refined in CORAL using restrictions in their attachment sites based on the experimental SAXS ZapE full curve.

Briefly, from the forward scattering intensity I(0) a MW of ~46 kDa was determined for a range of concentrations with a Radius of gyration (Rg) at around 31. Based on the indirect Fourier transform of the scattering curve the estimation of the maximum diameter of the particle was at ~100Å, indicative of an elongated particle (Fig. S3A, S3A and S3C). The pair distribution function and the ab initio models suggest the possibility of multiple folding domains (Fig. 1E). Although the Kratky Plot of the scattering data provided a clear maximum indicative of a folded particle (Fig. S3B), the presence of rigid domains in a particle with flexible loops cannot be ruled out. The fitting of the experimental SAXS curve to high s values took place using the ZapE sequence information. The position of the first rigid domain according to the second was evaluated by CORAL based on the SAXS curve while restrictions were used based to the non-modeled linker between the domains.The best fitting CORAL solution found (x = 1. 304) integrating fold recognition atomic models based on ZapE sequence and dummy residues to take into account not modelled residues by fitting the SAXS data up to smax = 0.4 Å−1.

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