The frozen grids were imaged in an FEI Polara 300 keV FEG transmission electron microscope (FEI Company, Hillsboro, OR) equipped with a Gatan energy filter (Gatan, Pleasanton, CA) and a Gatan K2 Summit direct detector (Gatan, Pleasanton, CA). Energy-filtered tilt-series of images of cells were collected automatically from −60° to +60° at 1° intervals using the UCSF Tomography data collection software51 (link) with a total dosage of 160 e−/Å2, a defocus of −6 µm and a pixel size of 3.9 Å. Data collection parameters are summarized in Supplementary Table 1 . The images were then binned by 2, aligned and contrast transfer function corrected using the IMOD software package52 (link). SIRT reconstructions were then produced using the TOMO3D program53 (link). TCPM structures on cell envelopes were located by visual inspection. Sub-tomogram averages with 2-fold symmetrization along the particle Y-axis were produced using the PEET program54 (link). Due to the flexibility observed between the OM- and IM-associated parts, binary masks were applied to cover one or the other for local alignment in PEET. The number of cryotomograms and particles used in the process are summarized in Supplementary Table 2 .
Fei polara 300 kev feg transmission electron microscope
Manufactured by Thermo Fisher Scientific
The FEI Polara 300 keV FEG transmission electron microscope is a high-performance imaging and analysis tool. It features a 300 kV field emission gun (FEG) electron source that provides high brightness and excellent resolution. The microscope is designed for advanced materials characterization and can be used for a variety of applications.
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2 protocols using fei polara 300 kev feg transmission electron microscope
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Cryo-Electron Tomography of Bacterial Cell Envelopes
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Cryo-Electron Tomography of Bacterial Cell Envelopes
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The frozen grids were imaged in an FEI Polara 300 keV FEG transmission electron microscope (FEI Company, Hillsboro, OR) equipped with a Gatan energy filter (Gatan, Pleasanton, CA) and a Gatan K2 Summit direct detector (Gatan, Pleasanton, CA). Energy-filtered tilt-series of images of cells were collected automatically from −60° to +60° at 1° intervals using the UCSF Tomography data collection software51 (link) with a total dosage of 160 e−/Å2, a defocus of −6 µm and a pixel size of 3.9 Å. Data collection parameters are summarized in Supplementary Table 1 . The images were then binned by 2, aligned and contrast transfer function corrected using the IMOD software package52 (link). SIRT reconstructions were then produced using the TOMO3D program53 (link). TCPM structures on cell envelopes were located by visual inspection. Sub-tomogram averages with 2-fold symmetrization along the particle Y-axis were produced using the PEET program54 (link). Due to the flexibility observed between the OM- and IM-associated parts, binary masks were applied to cover one or the other for local alignment in PEET. The number of cryotomograms and particles used in the process are summarized in Supplementary Table 2 .
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