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Title:Negative stain electron microscopy structure of Nup192
Authors:Sampathkumar P, Kim SJ, Upla P, Rice W, Phillips J, Pieper U, Bonanno JB, Fernandez-Martinez J, Ketaren NE, Matsui T, Stokes DL, Sauder JM, Burley SK, Sali A, Rout MP, Almo SC
Sample:Full-length Nup192
Method:Single particle reconstruction (26 angstroms resolution)
Red flagLatest update:2014-03-26
Sample
Sample name: Full-length Nup192
Oligomeric state: monomer
Experimental molecular weight of the sample: 0.192
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
1proteinSubunit of the Yeast Nuclear Pore Complex inner ring with weight 192 kDa0.1920.192monomertrueSaccharomyces cerevisiaeGO:0017056, GO:0006999, GO:0006913, GO:0005643, GO:0044611
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.40.0035 mg/mL20 mM HEPES, 300 mM NaCl, 2 mM MgCl2, 0.01% Tween 20, 0.01 mM DTT3 ul protein was added to grids. The sample was blotted and 3 uL 1% uranyl formate added. Uranyl formate was blotted and fresh stain added (three times). The sample was allowed to sit 30 seconds before final blot, then air-dried.200 mesh copper grid with thin carbon support, glow discharged 20s with Gatan Solarus 20s in H2/O2.
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
NONE% KNONE ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
JEOL 2100F200 kVFLOOD BEAMBRIGHT FIELD2 mm1500 nm2000 nm5000081911FIELD EMISSION GUNTVIPS TEMCAM-F224 (2k x 2k) mmObjective lens astigmatism was corrected at 250,000 times magnification
JEOL 2100F200 kVFLOOD BEAMBRIGHT FIELD2 mm1500 nm2000 nm5000081911FIELD EMISSION GUNTVIPS TEMCAM-F224 (2k x 2k) mmObjective lens astigmatism was corrected at 250,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
High tiltJEOL50° eV K K K mrad20 e/Å2Collected on 2028x2048 CCD, 24 um/pixel30-MAY-2012
High tiltJEOL50° eV K K K mrad20 e/Å2Collected on 2028x2048 CCD, 24 um/pixel. 2 sessions are listed; data collected on multiple days between these dates01-MAR-2012
Processing
Protocol:Random Conical Tilt
Software:Spider, Sparx, EMAN2, EMAN
CTF correction:phase flip each particle
Number of particles:3883
Number of class averages:23
Imposed symmetry:C1
Resolution by author:26 Å
Resolution method:FSC 0.5
Processing details:Initial map was calculated by merging 3 maps obtained from random conical tilt. This was then used for 8 rounds of reference-based refinement in SPIDER at a 10-degree angular increment. Particles were selected manually using jweb and boxer.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
40024 μm/pixel16
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
4IFQ rigid bodyCross-correlation 0.754ChimeraREALProtocol: Rigid Body. The domains were separately fitted by manual docking followed by "Fit in Map"using Chimera