EMD-1055

Mirrors
- RCSB (USA)
- EBI (UK)

Title:	Locking and unlocking of ribosomal motions.
Authors:	Valle M, Zavialov A, Sengupta J, Rawat U, Ehrenberg M, Frank J
Sample:	70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GDP-kirromycin.
Aggregation state:	Single particle (9 angstroms resolution)
Latest update:	2011-05-26

Sample

Sample name:

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

Components:

ID	Type	Name	Exp. MW (MDa)	Oligomeric details	Mutant	Organism	GO identifier	InterPro identifier	Virus identifier	Details
1	ribosome-prokaryote	-70S from E. coli
2	nucleic-acid	-fMet-tRNAfMet			false
3	nucleic-acid	-Phe-tRNAPhe			false
4	protein	-Elongation Factor Tu			false	n/a
5	nucleic-acid	-deacylated tRNA			false

Experiment

Sample preparation:

pH	Sample conc.	Details	Staining	Sample support details
7.5	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	Cryo-electron microscopy. No stain.	300 mesh Quantifoil R2/4

Vitrification:

Cryogen name	Humidity	Temp.	Instr.	Method	Time resolved	Details
ETHANE	%	90 K		two-face blotting for 1 second	ms

FEI TECNAI F20

200 kV

FLOOD BEAM

BRIGHT FIELD

2 mm

2000 nm

4000 nm

50000

49650

FIELD EMISSION GUN

Kodak SO163 film

corrected at 175,000

single tilt cryoholder

GATAN LIQUID NITROGEN

0°

90 K

mrad

15 e/Å²

Processing

Software:

Spider

CTF correction:

segregation in defocus groups and correction in volumes

Resolution by author:

9 Å

Resolution method:

FSC cut-off at 0.15

Unit cell:

Scanned images:

Num. images	Sampling size	OD range	Quant. bit number		Other details	Scanner
50	14 μm/pixel	1.0	16	link		ZEISS SCAI

Fitting:

PDB	Protocol	Target crit.	Software	B value	Fitting space	PDB chain	Details
1FNM 1FFK 1FJF	rigid body	correlation coeficient	O				Manual fitting in O.The docking of X-ray structures into the cryo-EM maps was made using O and the visualization was performed in IRIS Explorer. EF-G domains were taken from the Thermus thermophilus H573A mutant crystal structure in complex with GDP (PDB code: 1FNM). The coordinates of the ribosomal proteins and rRNAs were taken from published atomic structures (PDB code 1FFK for the 50S subunit; 1FJF for the 30S subunit and the docking was performed using the coordinates as rigid bodies.