EMD-2097 Experiments and Validation

Cryo-EM structure of the F420-reducing [NiFe]-hydrogenase from a methanogenic archaeon with bound substrate

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

Map parameters

Minimum density: -464.71
Maximum density: 1,390.65
Average density: 1.698
Standard deviation: 46.708
Recommended contour level: 212.00 (author)

Sample information

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

Validation information

Experimental information

 

Image processing

Applied symmetry: T
Software: EMAN2
Number of particles: 97000
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): 2.00
Nominal magnification: 59000.0
Defocus max (nm): 3,550.00
Defocus min (nm): 1,650.00
Holder model: OTHER
Acquisition
Number of images: 63
Scanner model: ZEISS SCAI

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.70
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