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Title:The bacterial DnaC helicase loader is a DnaB ring breaker
Authors:Arias-Palomo E, O'Shea VL, Hood IV, Berger JM
Sample:E. coli DnaB replicative helicase
Method:Single particle reconstruction (25 angstroms resolution)
Red flagLatest update:2013-04-24
Sample
Sample name: E. coli DnaB replicative helicase
Oligomeric state: Homohexamer
Theoretical molecular weight of the sample: 0.312
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
1proteinDnaB replicative helicase0.052hexamertrueEscherichia coliP0ACB0
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
8.5 mg/mL20 mM Tris-HCl pH 8.5, 200 mM NaCl, 5 % glycerol, 5 mM MgCl2, 1 mM beta-mercaptoethanol, and 1 mM ADP-BeF3Grids with adsorbed protein were floated on 2% w/v uranyl formate for 45 seconds400 mesh copper grid with thin carbon support, glow discharged for 20 seconds
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 12120 kVFLOOD BEAMBRIGHT FIELD6.3 mm700 nm1200 nm49000LAB6GENERIC TVIPS (4k x 4k) mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
SIDE ENTRY, EUCENTRIC°° eV297 K K K mrad25 e/Å221-JAN-2011
Processing
Protocol:Projection matching
Software:EMAN2, SPARX
CTF correction:Each micrograph
Number of particles:9617
Imposed symmetry:C3
Resolution by author:25 Å
Resolution method:FSC 0.5
Processing details:The particles were selected using DoG picker as available in APPION. The contrast transfer function of the microscope for each micrograph was estimated using CTFFIND3 and phase-flipped using SPIDER. DnaBC particles were subjected to a multi-model refinement as implemented in SPARX using the 3D averages obtained from the RCT reconstructions as initial references.
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails