EMD-2290 Experiments and Validation

First 3D model of wild type MjHSP16.5 at 60 degree Celsius by CryoEM.

Single particle reconstruction
Overview of EMD-2290
Sample name: wild type small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
Organism: Methanocaldococcus jannaschii
Related EM entries by publication: EMD-2288, EMD-2289, EMD-2293, EMD-2292, EMD-2291, EMD-2295, EMD-2294

Map parameters

Minimum density: -6.689
Maximum density: 8.135
Average density: 0.001
Standard deviation: 1.026
Recommended contour level: 1.73 (author)

Sample information

Sample name: wild type small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
Protein: MjHSP16.5

Validation information

Experimental information

 

Image processing

Applied symmetry: O
Software: EMAN1
Number of particles: 4842
Number of class averages: 193
Reconstruction protocol: projection matching
CTF correction: Each image
Details: The final reconstructed density map was further sharpened by application of an amplitude correction algorithm in the program BFACTOR. The particles were selected using an automatic selection program Gautomatch developed in Fei Sun lab (to be published). Octahedron symmetry were imposed during 3D reconstructing.

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 96000.0
Defocus max (nm): 3,000.00
Defocus min (nm): 2,000.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Number of images: 2119
Details: Electron micrograph exposures were made with the automatic collection package Leginon.

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.80
Buffer: 10mM HEPES, 100mM NaCl.
Specimen support: GIG holey grids (LifeTrust, China) were treated with a glow discharge machine (Master Plasmer)
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Protocol: The samples were first heated to 60 degree Celsius in the boiler, then 3.5 microlitre samples were added to the grid and blotted for 1.5 s with blot force 2 at 100% humidity;