EMD-2188 Experiments and Validation

human RNA polymerase II in complex with scAlu RNA

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

Map parameters

Recommended contour level: 0.05 (author)
Number of grid points: 146 × 146 × 146
Voxel size: 2.54 × 2.54 × 2.54 Å
Minimum density: -0.055
Maximum density: 0.167
Average density: -0.001
Standard deviation: 0.013

Sample information

Sample name: human RNA polymerase II in complex with scAlu RNA
Nucleic acid: scAlu
Proteins: Rpb1, Rpb12, Rpb11, Rpb10, Rpb9, Rpb8, Rpb7, Rpb6, Rpb5, Rpb2, Rpb4, Rpb3

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN2, FREALIGN
Number of particles: 25300
Reconstruction protocol: projection matching
CTF correction: each micrograph
Details: Projection matching using a low-pass filtered model of apo Pol II as the starting model

Fitting

Initial atomic model: 2B63
Used chains: A, B, C, D, E, F, G, H, I, J, K, L, R
Fitting software: SITUS
Fitting protocol: rigid body
Target criteria: correlation
Refinement space: REAL
Details: Protocol: rigid body

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: 50000.0
Defocus max (nm): 5,000.00
Defocus min (nm): 2,000.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 169
Scanner model: NIKON SUPER COOLSCAN 9000
Sampling interval (μm): 12.699999999999999

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
Staining procedure: cryo-EM
Specimen support: 400 mesh copper grids covered with a holey carbon film, thin carbon film floated on top
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 100
Protocol: blot for 6 sec, offset -2