JV9114

Description

Technical info

Tilt Series Date: 2017-07-08

Data Taken By: Debnath Ghosal

Species / Specimen: Legionella pneumophila

Strain: Philadelphia-1_Lp02

Tilt Series Settings: Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 100.0 eV/Ų, defocus: -6.0 μm, magnification: 26000x.

Microscope: Janelia Titan

Acquisition Software: UCSFTomo

Upload Method: pipeline

Processing Software Used: Raptor

Collaborators and Roles: Strain sent to us by Joseph P. Vogel, Washington University School of Medicine. Data collected and processed by Debnath Ghosal. Janelia Krios.

Purification / Growth Conditions / Treatment: Cells were grown in AYE liquid culture with following supplements: ferric nitrate, cysteine hydrochloride and thymidine. Scrape cells off a plate, dissolve in the media, adjust the cell density to 0.6 and make a dilution series. Start O/N culture with all of them and freeze cells next day from the one in early stationary phase that is OD ~ 3.0. This time cell density was 2.8

Sample Preparation: 6 grids were frozen for the JV9114 strain (2x concentrated culture); Vitrobot (MK iv) settings: 100% humidity, 4µl sample, Blot force 6, blot time 4.5 sec. Wait time 0 sec. Drain time 1 sec. Ethane/propane mix. Grid: Quantifoil Cu 2/2 200. 3 grids with extra-thick carbon (JV9114T) and three finder grids (JV9114F). Add gold to cells prior to freezing: 1ul of gold to 4 ul of cells, freeze.

Attached Files

area1-20170707-JV9114tomo0002.mrc, Tilt Series (Pixel Size 0.42 nm), 1.7 GB

area1-20170707-JV9114tomo0002_full.rec, Reconstruction (Pixel Size 1.68 nm), 445.4 MB