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Title:Cryo-EM of the Sec61-complex bound to an 80S-RNC engaged with a translocating LepT chain
Authors:Gogala M, Becker T, Beatrix B, Barrio-Garcia C, Berninghausen O, Beckmann R
Sample:Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating the translocating LepT-polypeptide
Method:Single particle reconstruction (7.4 angstroms resolution)
Red flagLatest update:2014-09-10
Sample
Sample name: Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating the translocating LepT-polypeptide
Oligomeric state: One Sec61 binds to one 80S ribosome
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
1proteinSec61-complex0.0699MonomerfalseCanis lupus familiarisData-subset resulted from computational sorting
2ribosome-eukaryote80S RibosomefalseTriticum aestivum
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.6 mg/mL30 mM Hepes/KOH 7.6, 10 mM Mg(OAc)2, 180 mM KOAC/HAc pH 7.6, 0.3 % Digitonin, 1 mM DTTQuantifoil grids pre-coated with 2 nm carbon on top
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE95% KFEI VITROBOT MARK IVBlot for 3 seconds before plunging ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TITAN KRIOS200 kVFLOOD BEAMBRIGHT FIELD2.7 mm1300 nm4000 nm148721FIELD EMISSION GUNTVIPS TEMCAM-F416 (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 mrad25 e/Å217-JUL-2011
Processing
Software:SPIDER, Signature, MAPPOS
CTF correction:On 3D-volume (SPIDER)
Number of particles:53248
Imposed symmetry:C1
Resolution by author:7.4 Å
Resolution method:FSC 0.5, semi-independent
Processing details:The particles were selected using SIGNATURE, classified using MAPPOS and processed with SPIDER.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
9805 μm/pixel16
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
2WWB flexibleChimera, Coot, MDFFREAL