EMD-5036

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Title:	Aminoacyl-tRNA-EF-Tu-GDP-kir ternary complex-bound E. coli 70S ribosome
Authors:	E.Villa,J.Sengupta,L.G.Trabuco,J.LeBarron,W.T.Baxter,T.R.Shaikh,R.A.Grassucci,P.Nissen,M.Ehrenberg,K.Schulten,J.Frank
Sample:	70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GDP-kirromycin.
Aggregation state:	Single particle (6.7 angstroms resolution)
Latest update:	2009-05-05

Sample

Sample name:

70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GDP-kirromycin.

Oligomeric state:

single particle

Components:

ID	Type	Name	Exp. MW (MDa)	Oligomeric details	Recombinant expression	Synthetic	Organism	GO identifier	InterPro identifier	Virus identifier	Details
1	ribosome-prokaryote	E. coli ribosome
2	nucleic-acid	fMet-tRNAfMet				false	Escherichia coli
3	nucleic-acid	Phe-tRNAPhe				false	Escherichia coli
4	nucleic-acid	tRNA				false	Escherichia coli
5	protein	Elongation factor Tu		monomer	true						EF-Tu bound to kirromycin and GDP

Experiment

Sample preparation:

pH	Sample conc.	Details	Staining	Sample support details
7.5	0.0768 mg/mL	5 mM potassium phosphate, 5 mM magnesium acetate, 5 mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 8 mM putrescine, 1 mM spermidine, and 1 mM dithioerythritol		Thin Carbon on 300 mesh Quantifoil R2/4

Vitrification:

Cryogen name	Humidity	Temp.	Instr.	Method	Time resolved	Details
ETHANE	90%	80 K	FEI VITROBOT	Blot 3 seconds before plunging	ms	Vitrification instrument: FEI vitrobot. Blot Offset at -1 mm

FEI POLARA 300

300 kV

FLOOD BEAM

BRIGHT FIELD

2.26 mm

1200 nm

4520 nm

59000

58279

FIELD EMISSION GUN

Kodak SO163 film

Objective lens astigmatism was corrected at 115,000 times magnification with fastscan ccd

FEI Polara Cartridge

OTHER

84 K

mrad

20 e/Å²

Low dose

31-AUG-2006

Processing

Software:

Spider

CTF correction:

Correction of reconstruction of each defocus group

Resolution by author:

6.7 Å

Resolution method:

FCS at 0.5 cut-off criterion, extrapolated to full dataset

Unit cell:

Scanned images:

Num. images	Sampling size	OD range	Quant. bit number		Other details	Scanner
304	7 μm/pixel	1.2	12	link		ZEISS SCAI

Fitting:

PDB	Protocol	Target crit.	Software	B value	Fitting space	PDB chain	Details
2I2U	MDFF	RMSD, cross correlation	MDFF		REAL		An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)
2I2V	MDFF	RMSD, cross correlation	MDFF		REAL		An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)
1OB2	MDFF	RMSD, cross correlation	MDFF		REAL		An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)