JV4404_delDot.Icm_Lp02

Description

Technical info

Tilt Series Date: 2015-12-11

Data Taken By: Debnath Ghosal

Species / Specimen: Legionella pneumophila

Strain: Philadelphia-1_Lp02

Tilt Series Settings: Single Axis, tilt range: (-50.0°, 60.0°), step: 1.0°, constant angular increment, dosage: 90.0 eV/Ų, defocus: -8.0 μm, magnification: 22500x.

Microscope: Caltech Polara

Acquisition Software: UCSFTomo

Upload Method: pipeline

Processing Software Used: Raptor, also manually processed 12 tomograms (and used SIRT) to check for presence of the T4SS basal-body

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 Caltech Polara.

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. Added gold before freezing.

Sample Preparation: 4 grids were frozen for JV4404 at OD= 2.9 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

JV44040014_part111_1.rec, Reconstruction (Pixel Size 1.92 nm), 445.4 MB

JV44040014.mrc, Tilt Series (Pixel Size 0.48 nm), 3.2 GB