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Title:Negative-stain electron microscopy of E. coli ClpB mutant E432A (BAP form bound to ClpP)
Authors:Carroni M, Kummer E, Oguchi Y, Clare DK, Wendler P, Sinning I, Kopp J, Mogk A, Bukau B, Saibil HR
Sample:ClpB E432A ATPgammaS. BAP variant bound to ClpP.
Method:Single particle reconstruction (18 angstroms resolution)
Red flagLatest update:2014-06-25
Sample
Sample name: ClpB E432A ATPgammaS. BAP variant bound to ClpP.
Oligomeric state: Homohexamer. (One homohexamer of BAP bound to one homoheptamer of ClpP)
Theoretical molecular weight of the sample: 0.5
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
1proteinClpB0.080.08HexamertrueEscherichia coliThe protein is engineered to bind to ClpP.
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.50.03 mg/mL20 mM Tris-HCl, pH 7.5, 20 mM KCl, 15 mM MgCl2, 1 mM DTT, 2 mM ATPgammaSprotein adsorbed on carbon coated grids pretreated with 0.01% poly lysine chains. Stained with 2% uranyl acetate for 1 minute.
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 F20200 kVFLOOD BEAMBRIGHT FIELD2 mm500 nm1200 nm5000068000FIELD EMISSION GUNGATAN K2 (4k x 4k) mmObjective lens astigmatism was corrected at 150,000 x 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 mrad20 e/Å206-JUN-2011
Processing
Protocol:angular reconstitution and projection matching
Software:IMAGIC, Spider
CTF correction:phase flipping entire frame
Number of particles:11570
Imposed symmetry:C6
Resolution by author:18 Å
Resolution method:FSC 0.5, semi-independent
Processing details:Starting models were generated by angular reconstitution and particle orientations were refined by projection matching in SPIDER. Only part of the molecule was refined in the alignment, but the final reconstruction includes the whole molecule.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
110 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1KHY rigid bodyChimeraREALFitting of separate domains was performed manually and locally optimised using Chimera. Known domain interfaces were used to guide the fit.
4CIU rigid bodyChimeraREALFitting of separate domains was performed manually and locally optimised using Chimera. Known domain interfaces were used to guide the fit.