SAXS Data Collection and Analyses

All SAXS data in this work was collected using SAXSpace instrument (Anton Paar GmbH, Austria). The instrument had a sealed tube X-ray source, a line collimated X-ray beam and a 1D CMOS Mythen detector (Dectris, Switzerland). The wavelength of X-rays was 0.154 nm and the sample to detector distance was 317.6 mm. Data was acquired from solutions of BlaC at concentrations of 4, 8 and 20 mg/ml to explore existence of any concentration dependent aggregation or inter-particle effect. For each concentration, the sample was exposed for 30 minutes (3 frames of 10 minutes each) at 10 °C in a thermostated quartz capillary with diameter of 1 mm. Thereafter, 100-fold molar excess of its known inhibitors clavulanic acid, sulbactam and tazobactam, and the inhibitors discovered in this study was added to BlaC and SAXS data was collected immediately for 40 minutes at intervals of 5 minutes. The data was calibrated for the beam position using SAXStreat software. The SAXSquant software was then used to subtract buffer contribution, set the usable q-range, and desmear the data using the beam profile. The SAXS data was further analyzed using the programs available in the ATSAS 2.7 suite of programs³³. The radius of gyration (R_G) was calculated on the basis of automated Guinier approximation using the PRIMUSQT integrated suite of programs³⁴. Same suite was used to compute the distance distribution function in auto-mode using the program GNOM which performs an Indirect Fourier transformation on the SAXS intensity profile³⁵. The Porod Exponent was calculated using the program Scatter3 (http://www.bioisis.net/scatter). The molecular mass of the scattering particles/protein molecules was calculated using the DATMOW program. Ab initio models were generated using the GASBOR program²⁵, and superimposed over the crystal structure and SREFLEX based model using the SUPCOMB program²⁶. For modelling the Apo state of BlaC, the data collected with concentration of 20 mg/ml was used, as it had the best Signal-to-Noise ratio. Using program SREFLEX²⁹, the crystal structure of BlaC (PDB ID: 2GDN) was subjected to normal mode analysis (NMA) to search for any models which may better agree with the SAXS data. The structures were visualized and the graphical representations were prepared using PyMOL v1.8³⁶. The IUCr guidelines published by Jacques et al. were followed for reporting all the SAXS data parameters³⁷.