Lp02_Legionella pneumophila

Description

Technical info

Tilt Series Date: 2015-10-29

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: 1.0°, constant angular increment, dosage: 100.0 eV/Ų, defocus: -8.0 μm, magnification: 26000x.

Microscope: Janelia Titan

Acquisition Software: UCSFTomo

Upload Method: pipeline

Processing Software Used: Raptor, also manually processed 25 tomograms (and used SIRT)for subtomogram averaging.

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: 4 grids were frozen for Lp02 at OD= 2.8 and another 4 grids for Lp02 at OD= 3.3Vitrobot (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

20151029_LP02_10059_part121_4.rec, Reconstruction (Pixel Size 1.68 nm), 430.6 MB

20151029_LP02_10059.mrc, Tilt Series (Pixel Size 0.42 nm), 3.3 GB

dga2015-10-29-59_slicer19128.png, A non-uniform distribution of ribosomes. . I am somewhat hesitant to call this a REZ . -andrew, 995.1 kB