Caltech Tomography Archive
Published 2018 | Version v1
Dataset Open

Vibrio cholerae SAD2095 induced

Creators

  • 1. ROR icon California Institute of Technology

Description

SAD2095 (T2SS and T4 competence pili) = rpoS*, pilA S81C, lacZ::lacIq, comEA-mCherry, ΔluxO, Ptac-tfoX, Ptac- recA, ∆TCP::ZeoR, ∆MSHA::CarbR, ∆CTX::KanR, ∆pilT::TmRAnkur: "For the taxonomy ID - unfortunately the strain we sent does not have a taxonomy ID. So, you can use the generic one for V. cholerae which is "666". The strain we are working with is related to N16961, but is certainly not the same strain, so not sure if you would want to indicate that somehow? Just for reference - the strain we sent is E7946 (although it is obviously heavily modified with a number of mutations). "

Technical info

Tilt Series Date: 2018-07-18

Data Taken By: Sara Weaver

Tilt Series Settings: Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 180.0 eV/Ų, defocus: -6.0 μm, magnification: 27500x.

Microscope: Caltech Polara

Acquisition Software: SerialEM

Upload Method: pipeline

Processing Software Used: Raptor

Collaborators and Roles: Strain from Ankur B Dalia ankdalia@indiana.edu, PhD, Assistant Professor, Indiana University. Cells grown, data collected by Sara Weaver

Purification / Growth Conditions / Treatment: Streaked stock on a plate. Grew up a single colony in 3 mL LB with shaking at 37˚C. The next day, subcultured 50uL of this overnight culture into 3mL of fresh LB+100uM IPTG + 20mM MgCl2 + 10mM CaCl2 and grow at 30˚C shaking for ~4 hours.

Sample Preparation: spun gold (100 µL 10 nm gold + 25 µL 5% BSA) at 15,000rpm for 20 minutes, removed supernatant. Resuspended gold beads in 20 µL liquid culture. Vitrobot RT and 95% humidity, 3µL of gold/sample slurry on carbon/copper Quantifoil Finder grids R2/2 (EmiTech glow discharge 50 mA 120 s) Blot force -6, blot time 3 s, no waiting time, no drain time. Plunged into ethane/propane mix.

swj2018-07-18-11_slicer19668.jpg: green + orange - T4P competence pili ? . pink - competence pilus basal body? . blue - flagellar filament swj2018-07-18-11_slicer19669.jpg: green - T4P competence pili ? . pink - competence pilus basal body? swj2018-07-18-11_slicer19670.jpg: green - T4P competence pili ? . pink - competence pilus basal body? . blue - flagellar filament swj2018-07-18-11_slicer19671.jpg: basal body? swj2018-07-18-11_slicer19672.jpg: membrane neck? swj2018-07-18-11_slicer19673.jpg: green - budding vesicle? . pink - object on extracellular surface? swj2018-07-18-11_slicer19674.jpg: densities in periplasm swj2018-07-18-11_slicer19675.jpg: densities in periplasm swj2018-07-18-11_slicer19676.jpg: densities in periplasm

Attached Files

20180718_tiltseries_11.mrc, Tilt Series (Pixel Size 0.39 nm), 2.8 GB

20180718_tiltseries_11_part98_3.rec, Reconstruction (Pixel Size 1.56 nm), 444.5 MB

Files

keyimg_swj2018-07-18-11.jpg

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Additional details

Identifiers

DOI
10.22002/m7q4s-rp331
TILT_SERIES_ID
swj2018-07-18-11

Funding

NIH
HHMI
Beckman Institute
Gordon and Betty Moore Foundation
Agouron Institute
John Templeton Foundation

Dates

Collected
2018-07-18
Created
2018-07-19
Updated
2018-07-19