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Title:Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
Authors:Sauerwald A, Sandin S, Cristofari G, Scheres SHW, Lingner J, Rhodes D
Sample:Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
Method:Single particle reconstruction (30 angstroms resolution)
Red flagLatest update:2013-04-17
Sample
Sample name: Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
Oligomeric state: Dimer
Experimental molecular weight of the sample: 0.670
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinTelomerase reverse transcriptase0.1270.67DimertrueHomo sapiensO14746GO:0000723IPR003545
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.60.01 mg/mL20 mM Tris, 150 mM KCl, 1 mM MgCl2Continuous 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.200 mesh carbon coated with thin carbon, glow discharged
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
NONE% KNONE ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI 12120 kVFLOOD BEAMBRIGHT FIELD mm1.0 nm1.5 nm4200042550TUNGSTEN HAIRPINKODAK SO-163 FILM mmCorrected at 100,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
SIDE ENTRY, EUCENTRIC°° eV K K K mrad10 e/Å2The films were developed in Kodak developer at full strength for 12 min.01-JAN-2011
Processing
Protocol:common lines
Software:EMAN2, XMIPP
Number of particles:20127
Number of class averages:100
Imposed symmetry:C1
Resolution by author:30 Å
Resolution method:FSC
Processing 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.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
4827 μm/pixel1.4The micrographs were compressed x4ZEISS SCAI
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails