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Title:Structural dynamics and inter-ring communication of the MecA-ClpC complex during active substrate unfolding and translocation revealed by cryo-EM
Authors:Liu J, Mei Z, Li N, Qi Y, Xu Y, Shi Y, Wang F, Lei J, Gao N
Sample:MecA-ClpC(E280A)
Method:Single particle reconstruction (9.0 angstroms resolution)
Red flagLatest update:2013-08-28
Sample
Sample name: MecA-ClpC(E280A)
Oligomeric state: Hexamer of ClpC with 6 bound MecA
Experimental molecular weight of the sample: 0.6
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
1proteinMecAtrueBacillus subtilis
2proteinClpCHexamertrueBacillus subtilis
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.50.03 mg/mL50mM kCl, 10mM Tris-HCL,2mM MgCl2, 2mM ATP
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100%90 KFEI VITROBOT MARK IVBlot for 2 seconds before plunging ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TITAN KRIOS300 kVFLOOD BEAMBRIGHT FIELD mm1500 nm3000 nm59000FIELD EMISSION GUNFEI EAGLE (4k x 4k) mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
FEI TITAN KRIOS AUTOGRID HOLDER°° eV K K K mrad20 e/Å209-MAR-2011
Processing
Protocol:Reference Projections
Software:SPIDER
CTF correction:each defocus group on 3D level
Number of particles:45514
Imposed symmetry:C6
Resolution by author:9.0 Å
Resolution method:FSC 0.5
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
994 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3PXI flexibleCross-correlationMDFFREALProtocol: Initial local fitting was done using Chimera and then MDFF was used for flexible fitting. ref: Trabuco, L.G., Villa, E., Mitra, K., Frank, J. and Schulten, K. (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics.