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Ribosome dynamics and tRNA movement as visualized by time-resolved electon cryomicroscopy

by single particle reconstruction, at 17 A resolution

Movie

Orientation:

#1: Surface view with section colored by density value, Surface level: 22, Image by UCSF CHIMERA

#2: Surface view colored by height, Surface level: 22, Image by UCSF CHIMERA

#3: Simplified surface model with fitted atomic model: PDB-3j5b, Image by Jmol

#4: Simplified surface model with fitted atomic model: PDB-3j5c, Image by Jmol

Entry
Summary
Database / IDEM DATA BANK (EMDB) / 1722
TitleRibosome dynamics and tRNA movement as visualized by time-resolved electon cryomicroscopy
MapSubstate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in intermediate post-translocation state (post2)
SampleSubstate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in intermediate post-translocation state (post2)
KeywordsRibosome, translation, translocation, tRNA
AuthorsFischer N, Konevega AL, Wintermeyer W, Rodnina MV, Stark H
DateDeposition: 2010-04-30, Header release: 2010-06-16, Map release: 2011-05-06, Last update: 2014-01-29
EMDB SitesEMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
MoviesMovie Page

#1: Surface view with section colored by density value, Surface level: 22, Image by UCSF CHIMERA

#2: Surface view colored by height, Surface level: 22, Image by UCSF CHIMERA

#3: Simplified surface model with fitted atomic model: PDB-3j5b, Image by Jmol

#4: Simplified surface model with fitted atomic model: PDB-3j5c, Image by Jmol

Supplemental images
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Article
Citation - Primary
ArticleNature, Vol. 466, Issue 7304, Page 329-33, Year 2010
TitleRibosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.
AuthorsNiels Fischer, Andrey L Konevega, Wolfgang Wintermeyer, Marina V Rodnina, Holger Stark
3D Electron Cryomicroscopy Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
KeywordsCryoelectron Microscopy, Escherichia coli, Kinetics, Models, Molecular, Molecular Conformation, Movement, Protein Biosynthesis, RNA, Transfer (genetics, 9014-25-9), Ribosome Subunits, Large, Bacterial (chemistry), Ribosome Subunits, Small, Bacterial (chemistry), Ribosomes (chemistry), Temperature, Thermodynamics, Time Factors
LinksDOI: 10.1038/nature09206, PubMed: 20631791
Map
Fileemd_1722.map.gz ( map file in CCP4 format, 8193 KB )
Projections & SlicesSize of images:
AxesZ (Sec.)Y (Row.)X (Col.)
128 pix
2.8 A/pix
= 358.4 A
128 pix
2.8 A/pix
= 358.4 A
128 pix
2.8 A/pix
= 358.4 A

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Density
Contour Level:22 (by author), 22 (movie #1):
Minimum - Maximum: -108.132 - 172.869
Average (Standard dev.): -1.06653 (18.6809)
Data TypeImage stored as Reals
Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin000
Limit127127127
Spacing128128128
Unit CellA= B= C: 358.4 A
Alpha=beta=gamma: 90 degrees
Pixel SpacingX= Y= Z: 2.8 A
CCP4 map header info
modeImage stored as Reals
A/pix X/Y/Z2.82.82.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
alpha/beta/gamma90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
start NC,NX/NR,NY/NS,NZ
NC,NX/NR,NY/NS,NZ
D min/max/mean-108.132172.869-1.067
Annotation DetailsSubstate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in intermediate post-translocation state (post2)
Supplement
Images
Images
Sample
NameSubstate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in intermediate post-translocation state (post2)
Number of Components4
Theoretical Mass2.5MDa
Component #1: ribosome-prokaryote - E. coli 70S
Scientific nameRibosome
Common NameE. coli 70S
Theoretical Mass2.5 MDa
Scientific Name of SpeciesEscherichia coli

NCBI taxonomy562
ProkaryoteALL
Recombinant expressionNo
Component #2: nucleic-acid - peptidyl tRNA
Scientific namefMetVal-tRNAVal
Common Namepeptidyl tRNA
Theoretical Mass0.025 MDa
Scientific Name of SpeciesEscherichia coli
NCBI taxonomy562
ClassT-RNA
StructureDOUBLE HELIX
SyntheticNo
Component #3: nucleic-acid - deacylated tRNA
Scientific nametRNAfMet
Common Namedeacylated tRNA
Theoretical Mass0.025 MDa
Scientific Name of SpeciesEscherichia coli
NCBI taxonomy562
ClassT-RNA
StructureDOUBLE HELIX
SyntheticNo
Component #4: nucleic-acid - mRNA
Scientific namem022 mRNA
Common NamemRNA
DetailsCoding sequence AUGGUU
Scientific Name of Speciessynthetic construct
NCBI taxonomy32630
StructureSINGLE STRANDED
SyntheticYes
ClassRNA
Experiment
Sample Preparation
Specimen Stateparticle
BufferDetails: 50 mM Tris-HCl, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2, 0.6 mM spermine, 0.4 mM spermidine
pH: 7.5
Vitrification
MethodManual blotting for about 2 seconds
Cryogen NameETHANE
Time Resolved StateSamples were vitrified at different time points along the reaction coordinate (1, 2, 5 and 20 minutes after addition of deacylated tRNAfMet to 70S-fMetVal-tRNAVal complexes)
DetailsVitrification instrument: Custom-built CEVS. Dew-point temperature (temperature on the grid) adjusted to 18 degrees C
Humidity75
InstrumentHOMEMADE PLUNGER
Temperature77 Kelvin
Imaging
MicroscopeFEI/PHILIPS CM200FEG
Electron Gun
Electron SourceFIELD EMISSION GUN
Accelerating Voltage160 kV
Electron Dose20 e/A**2
Illumination ModeSPOT SCAN
Lens
MagnificationNominal: 161000, Calibrated: 162740
AstigmatismObjective lens astigmatism was corrected at 200,000 times magnification
Nominal Cs2 mm
Imaging ModeBRIGHT FIELD
Defocus500 nm - 2000 nm
Specimen Holder
HolderEucentric
ModelGATAN LIQUID NITROGEN
Temperature77 K
Camera
DetectorGENERIC TVIPS (4k x 4k)
Processing
Methodsingle particle reconstruction
3D reconstruction
AlgorithmProjection matching
SoftwareIMAGIC, custom, Spider
CTF CorrectionLocal
DetailsFinal maps were calculated from 13 datasets acquired at different time points, computationally sorted into distinct substates
Resolution By Author17 A
Resolution MethodFSC 0.5
Single Particle
Number of Projections5656
Applied SymmetryC1 (asymmetric)
Atomic Model Fitting
Fitted Coordinate
PDB entry ID
Download
Data from EMDB
Header (meta data in XML format)emd-1722.xml (9 KB)
Map dataemd_1722.map.gz (7.4 MB)
Imagesimage1722.png (180.7 KB)
FTP directoryftp://ftp.pdbj.org/pub/emdb/structures/EMD-1722
Movie files
movie #1
.mp4 (H.264/MPEG-4 AVC format), 3.3 MB
.webm (WebM/VP8 format), 5.3 MB
Session file for UCSF-Chimera, 26.8 KB
movie #2
.mp4 (H.264/MPEG-4 AVC format), 3.1 MB
.webm (WebM/VP8 format), 4.6 MB
Session file for UCSF-Chimera, 27 KB
movie #3
.mp4 (H.264/MPEG-4 AVC format), 3.5 MB
.webm (WebM/VP8 format), 4.6 MB
movie #4
.mp4 (H.264/MPEG-4 AVC format), 3.6 MB
.webm (WebM/VP8 format), 4.9 MB