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Title:Cryo-electron microscopy structure of the Trypanosoma brucei 80S ribosome
Authors:Hashem Y, des Georges A, Fu J, Buss SN, Jossinet F, Jobe A, Zhang Q, Liao HY, Grassucci B, Bajaj C, Westhof E, Madison-Antenucci S, Frank J
Sample:Trypanosoma Brucei 80S Ribosome
Method:Single particle reconstruction (5.57 angstroms resolution)
Red flagLatest update:2013-02-27
Sample
Sample name: Trypanosoma Brucei 80S Ribosome
Oligomeric state: monomer
Theoretical molecular weight of the sample: 3.305
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-eukaryote80S Ribosome3.3falseTrypanosoma brucei
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.20.105 mg/mL20 mM Tris pH 7.2, 100mM MgCl2, 500 mM KCl, 5 mM beta-mercaptoethanol300 mesh Copper/Molbydenum holey carbon-coated Quantifoil 2/4 grid (Quantifoil Micro Tools GmbH) containing an additional continuous thin layer of carbon
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100%100 KFEI VITROBOT MARK IVWait 30 sec, Blot 6 seconds, plunge ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI POLARA 300300 kVFLOOD BEAMBRIGHT FIELD2.26 mm1500 nm4000 nm59000FIELD EMISSION GUNKODAK SO-163 FILM mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
SIDE ENTRY, EUCENTRIC°° eV K K K mrad25 e/Å201-JAN-2011
Processing
Protocol:conjugate gradients with regularization
Software:Spider
CTF correction:Phase-flip on each particle
Number of particles:164000
Imposed symmetry:C1
Resolution by author:5.57 Å
Resolution method:FSC 0.5
Processing details:Data were processed using SPIDER. The particles windows were automatically extracted from 1000 film-recorded micrographs and inspected manually. Standard SPIDER protocols for reference-based reconstruction, except that contrast transfer function (CTF) of the reconstructions was corrected by phase-flipping the particles using the defocus value estimated for each micrograph and a single reconstruction was obtained from the entire dataset using conjugate gradients with regularization (BP CG in SPIDER).
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
1000 μm/pixel32NIKON SUPER COOLSCAN 9000
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3U5B rigid bodycross correlationChimeraREALProtocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.
2XZM rigid bodycross correlationChimeraREALProtocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.
3IZR rigid bodycross correlationChimeraREALProtocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.