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Title:Cryo-EM structure of the 60S-Rei1 complex
Authors:Greber BJ, Boehringer D, Montellese C, Ban N
Sample:60S ribosomal subunit in complex with Rei1
Method:Single particle reconstruction (14.6 angstroms resolution)
Red flagLatest update:2012-12-12
Sample
Sample name: 60S ribosomal subunit in complex with Rei1
Oligomeric state: One monomer each of 60S and Rei1
Theoretical molecular weight of the sample: 2.4
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
1ribosome-eukaryote60S ribosomal subunit2.2falseSaccharomyces cerevisiae
2proteinRei10.046monomertrueSaccharomyces cerevisiaeIPR007087, IPR015880C-terminal His-tag
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
8.3 mg/mL20 mM Hepes-NaOH pH 8.3, 50 mM NaCl, 5 mM MgCl2, 5 mM beta-mercaptoethanolcryoQuantifoil holey carbon grid R2/1
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE%80 KHOMEMADE PLUNGERmanual blotting msVitrification instrument: manual plunger
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI 20200 kVFLOOD BEAMBRIGHT FIELD2.3 mm1300 nm6000 nm83000FIELD EMISSION GUNGATAN ULTRASCAN 4000 (4k x 4k) mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
eucentricGATAN LIQUID NITROGEN eV87 K K K mrad20 e/Å208-FEB-2012
Processing
Protocol:projection matching
Software:SPIDER
CTF correction:per frame
Number of particles:17415
Imposed symmetry:C1
Resolution by author:14.6 Å
Resolution method:FSC 0.5
Processing details:Fourier amplitudes of the reconstruction were enhanced using the SAXS curve from ribosomes; subsequently, the map was filtered in SPIDER using a butterworth low-pass filter with a filter midpoint of 0.22.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
15 μm/pixel16images were acquired using a 2 x 2 frame spot scan (per hole) using a serial EM script
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3U5H rigid bodycorrelationChimeraREALProtocol: rigid body
3U5I rigid bodycorrelationChimeraREALProtocol: rigid body