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- EMDB-6130: Negative stain random conical tilt reconstructions of E. coli rib... -

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Basic information

Entry
Database: EMDB / ID: 6130
TitleNegative stain random conical tilt reconstructions of E. coli ribosomal 30S subunit assembly intermediates
Map dataGroup V 30S assembly intermediate missing S3 density from wild-type E. coli (Figure 2F)
SampleGroup V 30S ribosomal subunit assembly intermediate missing S3 density from wild-type E. coli:
ribosome-prokaryote
KeywordsRibosome assembly / 30S subunit / Assembly intermediate
SourceEscherichia coli BW25113 (bacteria)
Methodsingle particle reconstruction / negative staining / 62 Å resolution
AuthorsSashital DG / Greeman CA / Lyumkis D / Potter CS / Carragher B / Williamson JR
CitationJournal: Elife / Year: 2014
Title: A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli.
Authors: Dipali G Sashital / Candacia A Greeman / Dmitry Lyumkis / Clinton S Potter / Bridget Carragher / James R Williamson
DateDeposition: Oct 8, 2014 / Header (metadata) release: Oct 22, 2014 / Map release: Oct 22, 2014 / Last update: Dec 3, 2014

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6130.map.gz (map file in CCP4 format, 43905 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
224 pix
2.05 Å/pix.
= 459.29 Å
224 pix
2.05 Å/pix.
= 459.29 Å
224 pix
2.05 Å/pix.
= 459.29 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.0504 Å
Density
Contour Level:1 (by author), 1 (movie #1):
Minimum - Maximum-1.31127524 - 3.44568634
Average (Standard dev.)-0.00159261 (0.22981738)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions224224224
Origin000
Limit223223223
Spacing224224224
CellA=B=C: 459.2896 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.05040178571432.05040178571432.0504017857143
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z459.290459.290459.290
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-1.3113.446-0.002

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Supplemental data

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Sample components

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Entire Group V 30S ribosomal subunit assembly intermediate missing S3 de...

EntireName: Group V 30S ribosomal subunit assembly intermediate missing S3 density from wild-type E. coli
Details: Group V particles from heterogeneous sample taken from the center of the 30S sucrose gradient peak
Number of components: 1
MassTheoretical: 500 kDa / Experimental: 820 kDa
Measured by: Sedimentation and calculation of MW of known components

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Component #1: ribosome-prokaryote, 30S assembly intermediate

Ribosome-prokaryoteName: 30S assembly intermediate
a.k.a: 30S ribosomal subunitProkaryotic small ribosomal subunit
Details: Particles from center of 30S sucrose gradient peak / Prokaryote: SSU 30S, PSR16s / Recombinant expression: No
MassTheoretical: 820 kDa
SourceSpecies: Escherichia coli BW25113 (bacteria)
External referencesGene Ontology: ribosome

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.015 mg/ml
Buffer solution: 20 mM Tris, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA, 6 mM 2-mercaptoethanol
pH: 7.5
Support filmC-flat grids (Protochips) with 2 micron diameter holes coated with a thin (2-5 nm) layer of continuous carbon support, plasma-cleaned for 5s
StainingNegative stain grids were prepared by applying the sample (3 uL) to the grid for 1 min, then blotting from the side to remove excess sample. The grid was washed immediately with 3 uL Buffer A, then blotted from the side. Concurrent with blotting, 3 uL of fresh 2% uranyl formate was applied to the grid, then blotted from the side. This step was repeated twice, then the grid was allowed to dry for at least 10 minutes.
VitrificationInstrument: NONE / Cryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI SPIRIT / Date: May 27, 2013
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 52000 X (nominal), 52000 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 52,000 times magnification using a live image of the power spectrum.
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 790 - 2250 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC / Tilt Angle: -50 - 0 deg. / Temperature: K ( 298 - K)
CameraDetector: TVIPS TEMCAM-F416 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 900
Details: 450 tilt pairs were collected at -50 and 0 degrees.

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 1 / Applied symmetry: C1 (asymmetric) / Number of projections: 248
Details: Image tilt pairs were collected (-50 and 0 degrees) and particle tilt pairs were identified and extracted as two separate stacks. The untilted stack was aligned and classified iteratively, and RCT volumes were created for a single class average by applying alignment parameters to the corresponding tilt pairs.
3D reconstructionAlgorithm: Random-conical tilt reconstruction / Software: Spider, Appion / CTF correction: Each micrograph / Details: RCT reconstruction / Resolution: 62 Å / Resolution method: FSC 0.5, semi-independent

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