EMD-2236 Experiments and Validation

Negative Staining Structure of Human Polycomb Repressive Complex 2 bound to the co-factor AEBP2

Single particle reconstruction
Overview of EMD-2236
Sample name: Negative Staining Reconstruction of Polycomb repressive Complex 2 bound to the co-factor AEBP2
Organism: Homo sapiens

Map parameters

Recommended contour level: 3.55 (author)
Number of grid points: 112 × 112 × 112
Voxel size: 3.14 × 3.14 × 3.14 Å
Minimum density: -6.361
Maximum density: 22.461
Average density: 0.00
Standard deviation: 1.00

Sample information

Sample name: Negative Staining Reconstruction of Polycomb repressive Complex 2 bound to the co-factor AEBP2
Proteins: RbAp48, Ezh2, Suz12, AEBP2, EED

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: Appion, Leginon, Spider, Imagic, EMAN2, SPARX
Number of particles: 39527
Number of class averages: 1000
Reconstruction protocol: Projection matching from RCT model
CTF correction: whole micrograph

Fitting

Fitting 1
Initial atomic model: 2QXV
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL
Details: Protocol: local rigid-body fitting algorithm
Fitting 2
Initial atomic model: 2YB8
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL
Details: Protocol: local rigid-body fitting algorithm

Imaging

Session
Microscope model: FEI TECNAI 20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Energy filter: FEI
Nominal CS (mm): 2.20
Nominal magnification: 80000.0
Defocus max (nm): 1,300.00
Defocus min (nm): 600.00
Tilt max (degrees): 0.00
Tilt min (degrees): 0.00
Holder model: OTHER
Acquisition
Number of images: 500

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.10
Buffer: 25 mM Hepes pH 7.5, 138 mM NaCl, 10% Glycerol, 0.05% NP40, 1 mM Tris(2-carboxyethyl) phosphine hydrochloride [TCEP]
Staining procedure: 2% Uranyl Formate
Specimen support: 400 mesh copper carbon support
Vitrification
Apparatus: NONE
Cryogen: NONE