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Title:Substrate-specific structural rearrangements of human Dicer
Authors:Taylor DW, Ma E, Shigematsu H, Cianfrocco MA, Noland CL, Nagayama K, Nogales E, Doudna JA, Wang HW
Sample:Human Dicer-PACT heterodimer in open conformation
Method:Single particle reconstruction (26 angstroms resolution)
Red flagLatest update:2013-06-19
Sample
Sample name: Human Dicer-PACT heterodimer in open conformation
Oligomeric state: monomer
Theoretical molecular weight of the sample: 0.260
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinEndoribonuclease DicermonomertrueHomo sapiensQ9UPY3GO:0031054IPR000999, IPR003100, IPR001650, IPR014001, IPR014720, IPR001159, IPR011545, IPR005034
2proteinInterferon-inducible double stranded RNA-dependent protein kinase activator AmonomertrueHomo sapiensO75569GO:0003725IPR001159, IPR014720
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.40.01 mg/mL20 mM HEPES, pH 7.5, 150 mM KCl, 3 mM EDTA, 1 mM DTT, and 2.5% glycerolAfter adsorption for 1 min, we stained the samples consecutively with three droplets of 2% (w/v) uranyl formate solution, blotted off the residual stain and air-dried the sample in a hood.glow-discharged holey carbon grids with a thin layer of carbon over the holes
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 FIELD5.2 mm400 nm1500 nm50000LAB6GENERIC TVIPS (4k x 4k) mmObjective lens astigmatism was corrected at 100,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
OTHER°° eV K K K mrad20 e/Å210-JUL-2012
Processing
Protocol:back projection
Software:EMAN2/SPARX, multi-model
CTF correction:each micrograph
Number of particles:10000
Imposed symmetry:C1
Resolution by author:26 Å
Resolution method:FSC 0.5
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
400 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails