EMD-2096 Experiments and Validation

Cryo-EM structure of the F420-reducing [NiFe]-hydrogenase from a methanogenic archaeon at near-atomic resolution

Single particle reconstruction
Overview of EMD-2096
Sample name: F420-reducing [NiFe] hydrogenase Frh
Organism: Methanothermobacter marburgensis
Fitted atomic model: 3zfs
Related in-frame EM entry: EMD-2097
Related EM entry by publication: EMD-2097

Map parameters

Minimum density: -506.05
Maximum density: 1,455.88
Average density: 3.11
Standard deviation: 46.9
Recommended contour level: 212 (author)

Sample information

Sample name: F420-reducing [NiFe] hydrogenase Frh
Protein: F420-reducing [NiFe] hydrogenase

Validation information

Experimental information

 

Image processing

Applied symmetry: T
Software: EMAN2
Number of particles: 84000
Number of class averages: 2134
CTF correction: each micrograph

Fitting

Initial atomic model: 2WPN
Used chains: A, B
Fitting software: chimera, coot
Fitting protocol: rigid body
Refinement space: REAL
Details: Protocol: A rigid body fit was done in Chimera and the model manually rebuilt in Coot.

Imaging

Session
Microscope model: FEI POLARA 300
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 15.0
Nominal CS (mm): 2
Nominal magnification: 59000.0
Defocus max (nm): 2,800
Defocus min (nm): 1,690
Holder model: OTHER
Acquisition
Number of images: 101
Scanner model: ZEISS SCAI

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.7
Buffer: 50mM Tris-HCl, 0.025mM FAD
Specimen support: freshly glow discharged Quantifoil holey grid (1 micrometer holes)
Vitrification
Apparatus: FEI VITROBOT MARK I
Cryogen: ETHANE
Humidity: 70
Protocol: blotting 2.5 seconds before plunging