Lp02_2nd

Description

Technical info

Tilt Series Date: 2016-01-15

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. This data was collected at the Janelia Krios.

Purification / Growth Conditions / Treatment: Lp02 is thymidine auxotroph of the parental strain Lp01 originating from Philadelphia-1 strain. 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. Added gold before freezing.

Sample Preparation: Grids were stored in Janelia from previous imaging. See previous Lp02 sample prep. 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.

dga2016-01-15-50_slicer16667.jpg: Cell punctured | Releasing vesicles | lots of electron dense stuff in the periplasm | dga2016-01-15-50_slicer16668.jpg: Cell punctured | Releasing vesicles | lots of electron dense stuff in the periplasm | dga2016-01-15-50_slicer16669.jpg: Cell punctured | Releasing vesicles | lots of electron dense stuff in the periplasm | Dot/Icm

Attached Files

tomo_area1000100010028.mrc, Tilt Series (Pixel Size 0.42 nm), 2.2 GB

tomo_area1000100010028_full.rec, Reconstruction (Pixel Size 1.68 nm), 430.6 MB