EMD-2294 Experiments and Validation

First 3D model of the MjHSP16.5 mutant R107G at 60 degree Celsius by CryoEM.

Single particle reconstruction
Overview of EMD-2294
Sample name: R107G mutation of 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-2290, EMD-2295

Map parameters

Minimum density: -6.19
Maximum density: 8.44
Average density: 0
Standard deviation: 1.02
Recommended contour level: 2.12 (author)

Sample information

Sample name: R107G mutation of small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
Protein: MjHSP16.5 R107G mutation

Validation information

Experimental information

 

Image processing

Applied symmetry: O
Software: EMAN1
Number of particles: 4482
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): 2.7
Nominal magnification: 96000.0
Defocus max (nm): 3,000
Defocus min (nm): 2,000
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Number of images: 1308
Details: Electron micrograph exposures were made with the automatic collection package Leginon.

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.8
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 heated to 60 degree Celsius in boiler, then 3.5 microliter samples were added to the grid and blotted for 2 s with blot force 2 at 90% humidity.