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Title:Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104.
Authors:Petra Wendler, James Shorter, Celia Plisson,
Sample:Yeast Hsp104 N728A ATPgS
Aggregation state:Single particle (13 angstroms resolution)
Red flagLatest update:2011-05-26
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bullet_white.gif  Summary
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bullet.png Experimental details
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Sample
Sample name: Yeast Hsp104 N728A ATPgS
Oligomeric state: hexamer
Theoretical molecular weight of the sample: 0.612
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism GO identifier InterPro identifier Virus identifier Details
1proteinHsp104 N728A0.102HexamertrueSaccharomyces cerevisiae
2ligandATPgammaSfalse
Experiment
Sample preparation:
pHSample conc.DetailsStainingSample support details
7.50.3 mg/mL20 mM HEPES pH 7.5, 20 mM NaCl, 10 mM MgCl2, 1 mM DTT, 2mM ATPgS300 mesh copper grid- holey carbon film
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE% KHOMEMADE PLUNGERThe grids were blotted for 2-3 sec and immediately plunged into liquid ethane msVitrification instrument: home made
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20200 kVFLOOD BEAMBRIGHT FIELD2 mm1400 nm3900 nm5000050000FIELD EMISSION GUNKodak SO163 film mmobjective lens astigmatism was corrected for at

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
single tilt cryoGATAN LIQUID NITROGEN°° eV77 K77 K85 K mrad15 e/Å22005-03-17
Processing
Software:Imagic, Spider
CTF correction:phase flipping, each particle
Resolution by author:13 Å
Resolution method:FSC at 0.5 cut-off
Unit cell:
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
357 μm/pixel18linkZEISS SCAI
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
The domains were manually fitted as rigid bodies using Pymol. Automated fitting in Chimera optimised fit for NBD2.
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