EMD-2194 Experiments and Validation

human RNA Polymerase II

Single particle reconstruction
Overview of EMD-2194
Sample name: human Pol II
Organism: Homo sapiens
Related EM entries by publication: EMD-2188, EMD-2189, EMD-2190, EMD-2191, EMD-2192, EMD-2193

Map parameters

Recommended contour level: 0.139 (author)
Number of grid points: 128 × 128 × 128
Voxel size: 3.01 × 3.01 × 3.01 Å
Minimum density: -0.12
Maximum density: 0.357
Average density: -0.006
Standard deviation: 0.03

Sample information

Sample name: human Pol II
Proteins: Rpb12, Rpb11, Rpb10, Rpb9, Rpb8, Rpb7, Rpb6, Rpb5, Rpb4, Rpb3, Rpb2, Rpb1

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN2, FREALIGN
Number of particles: 8905
Reconstruction protocol: projection matching
CTF correction: whole micrograph
Details: the data set was sorted into three different conformations using an automated multi-model approach; the initial reconstructions were used as starting models for multi-model projection matching in EMAN2, followed by final refinement in FREALIGN

Imaging

Session
Microscope model: FEI TECNAI 20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.20
Nominal magnification: 80000.0
Defocus max (nm): 3,000.00
Defocus min (nm): 1,200.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 972
Sampling interval (μm): 1.5
Details: data collected with leginon

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.031
Buffer: 10 mM Tris pH 7.9, 10 mM HEPES pH 8.0, 4 mM MgCl2, 50 mM KCl, 0.05% NP-40, 1 mM DTT, 0.1% trehalose
Specimen support: 400 mesh C-flat grids (Protochips Inc.) with a thin carbon film floated on top
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 100
Protocol: 6 sec blot, offset -2