Structural insights into Pseudomonas aeruginosa lysine-specific uptake mechanism for extremely low pH regulation

The dataset denotes the cryo-EM maps and model files for the cryo-EM structure of lysine-specific permease bound to L-lysine and in complex with a nanobody.
Electron microscopy sample preparation and data acquisition:
The cryo-EM grids were prepared by applying 3 μL of the LysP-Nb5755 complex protein at 2 mg/mL in buffer containing 100 mM NaCl, 20 mM Tris-HCl pH 7.5, 0.03 %(w/v) DDM and 10 mM L-lysine, to a glow-discharged Quantifoil R1.2/1.3 200/300-mesh copper/gold holey carbon grid (QUANTIFOIL, Micro Tools GmbH, Germany) and blotted for 3.0 s under conditions of 100% relative humidity and 4°C before being plunged into liquid ethane using a Mark IV Vitrobot (FEI). Grids were screened in a 200 keV Talos Arctica microscope (Thermo Fisher) at Uppsala University, Sweden and the Membrane Protein Lab, Diamond Light Source Ltd., UK cryo-EM imaging facilities. Micrographs were acquired on a Titan Krios microscope (FEI) operated at 300 kV with a K2 Summit direct electron detector (Gatan) at the cryo-EM Swedish National Facility in Stockholm. SerialEM software was used for automated data collection following standard procedures. A calibrated magnification of ×130,000 was used for imaging, yielding a pixel size of 0.67 Å on images. The defocus range was set from −0.8 to −2.4 μm. Each micrograph was dose-fractionated to 50 frames under a dose rate of 7 - 8 electrons.pixel-1.s-1, with a total exposure time of 8 s, resulting in a total dose of ~80 electrons.Å-2.
Cryo-EM data processing, model building, and structure analysis:
The data set was processed using CryoSPARC64. The dose fractionated movie frames were aligned using “patch motion correction”, contrast transfer function (CTF) were estimated using “Patch CTF estimation”, the particles were picked using automated blob picker and extracted by binning 6 times in cryoSPARC live. Afterwards, several rounds of 2D classifications were performed. Finally, good particles were selected and extracted by binning equal to 1.0 Å.pixiel-1. 177,424 particles were further used for ab initio maps and heterogeneous refinements. After refinement, 145,063 particles were selected. Several rounds of non-uniform refinement and local refinement were performed. To remove bad particles, further phase randomized heterogeneous refinement was performed with low-pass filter 8 Å, 20 Å and 40 Å. The 78,459 particles were selected and local refinement with a tight mask was performed. The resolution reached 3.68 Å at the gold standard FSC resolution value of 0.143. The local resolution was calculated in CryoSPARC64.
An AlphaFold 265 model of LysP was automatically fitted into the cryo-EM density map of our inward-facing state. Iterative model building and real space refinement were performed using COOT and PHENIX.refine.