WT tomogram data

Description

Technical info

Tilt Series Date: 2020-12-28

Data Taken By: Rachel Rosenzweig

Species / Specimen: Caulobacter crescentus

Strain: NA1000

Tilt Series Settings: Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 180.0 eV/Ų, defocus: -8.0 μm, magnification: 26000x.

Microscope: Caltech Titan Krios

Acquisition Software: Serial EM

Upload Method: pipeline

Processing Software Used: RAPTOR

Collaborators and Roles: Patrick Viollier

Purification / Growth Conditions / Treatment: Inoculated a single colony from a 2% M2G agar plate in 5mL M2G at 30C, 220rpm for 24hr. Diluted each 5mL culture in 15mL M2G at 30C, 220rpm for 3hrs to OD600 0.7. Transferred to a conical centrifuge tube. Centrifuged at 6,400xg at 4C for 10min. (Media and tubes now on ice). Aspirated supernatant, resuspended with 1mL cold 1X M2. Transferred to a new 2mL microcentrifuge tube. Centrifuged at 15,000xg at 4C for 3min. Aspirated supernatant, resuspended with 900uL cold 1X M2. Added and mixed 900uL cold Percoll® [Sigma-Aldrich #P4937]. Centrifuged at 15,000xg at 4C for 20min. Aspirated top stalked cells band, collected bottom swarmer band. Transferred to a new 1.5mL microcentrifuge tube. Washed cells by adding 1mL cold 1X M2. Centrifuged at 15,000xg at 4C for 3min. (**Repeated wash 3x). Aspirated supernatant to leave 20uL cell solution resuspended.

Sample Preparation: Mixed 1mL 10nm Au with 250uL 5% BSA. Vortexed. Centrifuged at 18,000xg for 30min. Aspirated supernatant to leave 20uL Au solution resuspended. Mixed 1:1 ratio of Au:Cells. Vitrobot 3uL of solution @ RT with 100% humidity. Blot force: 2. Blot time: 4s. Quantifoil R3.5/1 200 Mesh grids

Attached Files

Caulobacter325_movies.mrc, Tilt Series (Pixel Size 0.33 nm), 2.8 GB

Caulobacter325_movies_full.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB

Caulobacter325.mrc.mdoc, 42.7 kB