EMD-2310 Experiments and Validation

Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain

Single particle reconstruction
Overview of EMD-2310
Sample name: Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
Organism: Homo sapiens
Related EM entry by publication: EMD-2311, EMD-2312

Map parameters

Recommended contour level: 0.097 (author)
Number of grid points: 48 × 28 × 28
Voxel size: 6.60 × 6.60 × 6.60 Å
Minimum density: -0.206
Maximum density: 0.418
Average density: -0.013
Standard deviation: 0.084

Sample information

Sample name: Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
Protein: Telomerase reverse transcriptase

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN2, XMIPP
Number of particles: 20127
Number of class averages: 100
Reconstruction protocol: common lines
Details: 3D structure was refined using the low-resolution subtomogram average as a reference model for 3D single particle refinement with EMAN2. Distinct conformations were further classified by three-dimensional maximum-likelihood analysis in Fourier space (MLF3D). Density map calculated from a sub-population of 3,659 particles. The absolute hand and the overall correctness of the dimer structure were assessed using a modified version of tilt-pair validation method. Electron tomography was used to calculate a low-resolution reference model. Distinct conformations were further classified by three-dimensional maximum-likelihood analysis in Fourier space (MLF3D).Density map calculated from a sub-population of 3,659 particles.

Imaging

Session
Microscope model: FEI TECNAI 12
Electron source: TUNGSTEN HAIRPIN
Electron dose (e/A**2): 10.0
Nominal magnification: 42000.0
Defocus max (nm): 1.50
Defocus min (nm): 1.00
Holder model: SIDE ENTRY, EUCENTRIC
Details: The films were developed in Kodak developer at full strength for 12 min.
Acquisition
Number of images: 482
Scanner model: ZEISS SCAI
Sampling interval (μm): 7.0
Details: The micrographs were compressed x4

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.01
Buffer: 20 mM Tris, 150 mM KCl, 1 mM MgCl2
Staining procedure: Continuous carbon-coated grids were freshly prepared and glow-discharged before use. 13 microl of telomerase sample (8-10 nM) were deposited on the grid for 15-30 minutes, blotted with filter paper and negatively stained with 2 drops of 1-2% (w/v) uranyl acetate solution.
Specimen support: 200 mesh carbon coated with thin carbon, glow discharged
Vitrification
Apparatus: NONE
Cryogen: NONE