JV3559_Janelia

Description

Technical info

Tilt Series Date: 2017-01-08

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: 1.50°, 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 at F30, Caltech.

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 ~3.4

Sample Preparation: 8 grids were frozen for the JV3559 strain at OD~ 3.4, 4 grids at density x and another 4 at density 2x. Vitrobot (MK iv) settings: 100% humidity, 4µl sample, Blot force 6, blot time 3 sec. Wait time 0 sec. Drain time 1 sec. Ethane/propane mix. Grid: Quantifoil Cu 2/2 200. Add gold to cells prior to freezing: 1ul of gold to 4 ul of cells, freeze.

Attached Files

JV3559_20170108a_area20006_part81_1.rec, Reconstruction (Pixel Size 1.68 nm), 445.4 MB

JV3559_20170108a_area20006.mrc, Tilt Series (Pixel Size 0.42 nm), 2.3 GB

dga2017-01-08-102_slicer17816.png, Have seen this compartment many many times in Legionella!, 725.4 kB