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Time-resolved Cryo Electron Microscopy of ribosome subunit association

by single particle reconstruction, at 9.7 A resolution

Movie

Orientation:

#1: Surface view with section colored by density value, Surface level: 0.04, Imaged by UCSF CHIMERA

#2: Surface view colored by height, Surface level: 0.04, Imaged by UCSF CHIMERA

Entry
Summary
Database / IDEM DATA BANK (EMDB) / 2976
TitleTime-resolved Cryo Electron Microscopy of ribosome subunit association
MapReconstruction of E. Coli naked 70S ribosome in non-rotated (NR) conformation
SampleE. Coli 70S Ribosome
Keywordstime-resolved, cryo-EM, mixing-spraying, ribosome subunit association, structural dynamics
AuthorsChen B, Kaledhonkar S, Sun M, Shen B, Lu Z, Barnard D, Lu T, Gonzalez Jr R, Frank J
DateDeposition: 2015-04-07, Header release: 2015-06-17, Map release: 2015-06-17, Last update: 2015-06-17
EMDB SitesEMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
MoviesMovie Page

#1: Surface view with section colored by density value, Surface level: 0.04, Imaged by UCSF CHIMERA

#2: Surface view colored by height, Surface level: 0.04, Imaged by UCSF CHIMERA

Supplemental images
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Article
Citation - Primary
ArticleStructure, Vol. 23, Issue 6, Page 1097-1105, Year 2015
TitleStructural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy.
AuthorsBo Chen, Sandip Kaledhonkar, Ming Sun, Bingxin Shen, Zonghuan Lu, David Barnard, Toh-Ming Lu, Ruben L Gonzalez, Joachim Frank
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Wadsworth Center, Albany, New York State Department of Health, Albany, NY 12201, USA.
Department of Chemistry, Columbia University, New York, NY 10027, USA.
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032, USA; Department of Biological Sciences, Columbia University, New York, NY 10027, USA; Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
KeywordsCryoelectron Microscopy (methods), Escherichia coli (chemistry), Models, Molecular, Protein Conformation, Ribosome Subunits (chemistry), Time Factors
LinksPubMed: 26004440, PII: S0969-2126(15)00135-5, DOI: 10.1016/j.str.2015.04.007, PMC: PMC4456197
Map
Fileemd_2976.map.gz ( map file in CCP4 format, 16001 KB )
Projections & SlicesSize of images:
AxesZ (Sec.)Y (Row.)X (Col.)
160 pix
1 A/pix
= 160. A
160 pix
1 A/pix
= 160. A
160 pix
1 A/pix
= 160. A

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Density
Contour Level:0.04 (by author), 0.04 (movie #1):
Minimum - Maximum: -0.09787601 - 0.19526787
Average (Standard dev.): 0.00143119 (0.02871864)
Data TypeImage stored as Reals
Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions160160160
Origin000
Limit159159159
Spacing160160160
Unit CellA= B= C: 160 A
Alpha=beta=gamma: 90 degrees
Pixel Spacing
XYZ
EMDB info.111
CCP4 map header111
EM Navigator Movie #12.252.252.25
CCP4 map header info
modeImage stored as Reals
A/pix X/Y/Z111
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z160.000160.000160.000
alpha/beta/gamma90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
start NC,NX/NR,NY/NS,NZ
NC,NX/NR,NY/NS,NZ
D min/max/mean-0.0980.1950.001
Annotation DetailsReconstruction of E. Coli naked 70S ribosome in non-rotated (NR) conformation
Supplement
Images
Images
Sample
NameE. Coli 70S Ribosome
Number of Components1
Component #1: ribosome-prokaryote - Escherichia coli 70S ribosome
Scientific nameEscherichia coli 70S ribosome
Scientific Name of SpeciesEscherichia coli

NCBI taxonomy562
StrainMRE600
ProkaryoteLSU 50S, SSU 30S
Recombinant expressionNo
Experiment
Sample Preparation
Specimen Stateparticle
Specimen Support DetailsQuantifoil R2/2 300 mesh copper grid with thin carbon sipport
BufferDetails: 25 mM Tris-HCl, 60 mM NH4Cl, 5 mM 2-mercaptoethanol, 3.5 mM MgCl2
pH: 7.6
Vitrification
Cryogen NameETHANE
Time Resolved StateVitrified after spraying
DetailsEqual volume of 1.2 microM 30S and 0.6 microM 50S (final concentration after mixing) were injected into the mixing-spraying device each at flow rate of 3 microL/s. The computer-controlled plunging device was purchased from Dr. Howard White (Eastern Virginia Medical School, VA).
Humidity80
InstrumentOTHER
Temperature80 Kelvin
Imaging
MicroscopeFEI TECNAI F20
Date13-SEP-2013
DetailsLow dose, Data was collected over two years time
Electron Gun
Electron SourceFIELD EMISSION GUN
Accelerating Voltage200 kV
Electron Dose17 e/A**2
Illumination ModeFLOOD BEAM
Lens
MagnificationNominal: 50000, Calibrated: 66318
Nominal Cs2 mm
Imaging ModeBRIGHT FIELD
Defocus2000 nm - 4000 nm
Specimen Holder
ModelGATAN LIQUID NITROGEN
Temperature80 K
Camera
DetectorGATAN ULTRASCAN 4000 (4k x 4k)
Image Acquisition
#1
Number of Digital Images3402
Processing
Methodsingle particle reconstruction
3D reconstruction
SoftwareArachnid, RELION, SPIDER
CTF Correctioneach Micrograph
Resolution By Author9.7 A
Resolution MethodFSC 0.143, gold-standard
Single Particle
Applied SymmetryC1 (asymmetric)
Number of Projections39678
DetailsThe partciles were selected with Autopicker (Langlois et al., 2014), and 3D classification and reconstruction with RELION
Download
Data from EMDB
Header (meta data in XML format)emd-2976.xml (7.3 KB)
Map dataemd_2976.map.gz (14.4 MB)
Imagesemd_2976.png (304.2 KB)
FTP directoryftp://ftp.pdbj.org/pub/emdb/structures/EMD-2976
Movie files
movie #1
.mp4 (H.264/MPEG-4 AVC format), 3.5 MB
.webm (WebM/VP8 format), 3.5 MB
Session file for UCSF-Chimera, 26.9 KB
movie #2
.mp4 (H.264/MPEG-4 AVC format), 3.2 MB
.webm (WebM/VP8 format), 3 MB
Session file for UCSF-Chimera, 27 KB