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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
Method:Single particle reconstruction (26 angstroms resolution)
Red flagLatest update:2013-06-19
Sample
Sample name: Human Dicer
Oligomeric state: monomer
Theoretical molecular weight of the sample: 0.220
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
1proteinEndoribonuclease Dicer0.220monomertrueHomo sapiensQ9UPY3GO:0031054IPR000999, IPR003100, IPR001650, IPR014001, IPR014720, IPR001159, IPR011545, IPR005034
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.40.05 mg/mL20 mM HEPES, pH 7.5, 150 mM KCl, 3 mM EDTA, 1 mM DTT, and 2.5% glycerolglow-discharged Quantifoil R 1.2/1.3 MO 200 mesh holey carbon grids
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100% KFEI VITROBOT MARK IVThe samples were automatically blotted for 4-5 s at -2.5 mm offset before plunging. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
JEOL 3100FFC300 kVFLOOD BEAMBRIGHT FIELD3.7 mm0 nm0 nm60000100000FIELD EMISSION GUNGENERIC GATAN (2k x 2k) 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
Liquid helium cooled stage maintained at 55 KJEOL°°JEOL0-20 eV55 K45 K60 K mrad20 e/Å210-AUG-2009
Processing
Protocol:back projection
Software:SPIDER
CTF correction:each micrograph
Number of particles:4800
Imposed symmetry:C1
Resolution by author:26 Å
Resolution method:FSC 0.5
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
800 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails