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Title:Single particle cryo-microscopy reconstruction of the isolated Manduca sexta V1 domain
Authors:Muench SP, Scheres SHW, Huss M, Phillips C, Vitavska O, Wieczorek H, Trinick J, Harrison MA
Sample:M. sexta isolated V1 domain without subunit C
Method:Single particle reconstruction (20 angstroms resolution)
Related entries in frame:EMD-2317
Red flagLatest update:2014-01-15
Sample
Sample name: M. sexta isolated V1 domain without subunit C
Oligomeric state: One V1 bound to a monomer of subunit C
Theoretical molecular weight of the sample: 0.65
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinVacuolar ATPase isolated V1 domain0.653A,B,E,G subunits and 1 D,F,H subunitfalseManduca sextaNo C subunit
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
8.11 mg/mL150mM NaCl, 20mM Tris-HCL, 0.01% C12E10negative stain data was collected using 1% w/v uranyl acetate for 1 minute.UV glow discharged grids, 400 mesh
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100%120 KFEI VITROBOT MARK IVGrids were blotted for 6 seconds with a 6 second drain time ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20200 kVFLOOD BEAMBRIGHT FIELD2.0 mm1 nm3.5 nm5000069000FIELD EMISSION GUNGENERIC GATAN (4k x 4k) mmAstigmatism corrected for each image in focus mode at 100,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Nitrogen cooled 3350 holderGATAN LIQUID NITROGEN°° eV100 K90 K110 K mrad16 e/Å2FEI Low dose mode14-APR-2011
Processing
Protocol:Maximum Likelihood
Software:Relion
CTF correction:Relion
Number of particles:16500
Imposed symmetry:C1
Resolution by author:20 Å
Resolution method:Gold standard FSC curve, gold-standard
Processing details:Maximum likelihood in Relion using the MLF3D protocol. The particles were handpicked in BOXER.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
μm/pixelAll collected on Gatan 4K x 4K CCD
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3A5D rigid bodybest fitChimeraREALThe domains were fitted using Chimera