EMD-5850 Experiments and Validation

Cryo-electron tomography of Chlamydomonas axoneme labeled with streptavidin (RSP6 N-terminus tag)

Subtomogram averaging
Overview of EMD-5850
Sample name: Chlamydomonas axoneme, RSP6 BCCP N-terminus tag
Organism: Chlamydomonas reinhardtii

Map parameters

Minimum density: 0
Maximum density: 39,462.37
Average density: 10,783.84
Standard deviation: 14,643
Recommended contour level: 34,398 (author)

Sample information

Sample name: Chlamydomonas axoneme, RSP6 BCCP N-terminus tag
Cellular component: Axoneme

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: PEET, RUBY-HELIX
Number of subtomograms averaged: 502

Imaging

Session
Microscope model: JEOL 3100FFC
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 90.0
Energy filter: JEOL Omega
Energy window (eV): 0
Nominal CS (mm): 2.2
Nominal magnification: 15000.0
Tilt max (degrees): 60.5
Tilt min (degrees): -60.5
Holder model: GATAN LIQUID NITROGEN

Specimen

Preparation
Specimen state: filament
Specimen concentration (mg/mL): 0.01
Buffer: 30 mM Hepes-NaOH pH 7.2, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, and 50 mM NaCl
Specimen support: 300 mesh copper grid with home-made holey carbon support
Vitrification
Apparatus: LEICA EM GP
Cryogen: ETHANE
Humidity: 90
Protocol: Blot for 5 seconds before plunging