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- EMDB-4082: Structure of bacterial 30S-IF1-IF2-IF3-mRNA-tRNA translation pre-... -

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Basic information

Entry
Database: EMDB / ID: EMD-4082
TitleStructure of bacterial 30S-IF1-IF2-IF3-mRNA-tRNA translation pre-initiation complex(state-II)
Map data
Sample
  • Complex: 30S-IF1-IF2-IF3-mRNA-tRNA pre-initiation complex (state-II)
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.3 Å
AuthorsHussain T / Llacer JL / Wimberly BT / Ramakrishnan V
CitationJournal: Cell / Year: 2016
Title: Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation.
Authors: Tanweer Hussain / Jose L Llácer / Brian T Wimberly / Jeffrey S Kieft / V Ramakrishnan /
Abstract: In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 ...In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition.
History
DepositionAug 1, 2016-
Header (metadata) releaseAug 10, 2016-
Map releaseOct 5, 2016-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4082.png

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Map

FileDownload / File: emd_4082.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 260 pix.
= 348.4 Å		1.34 Å/pix.
x 260 pix.
= 348.4 Å		1.34 Å/pix.
x 260 pix.
= 348.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.08887035 - 0.20115758
Average (Standard dev.)0.0011401811 (±0.015841644)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 348.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z348.400348.400348.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.0890.2010.001

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Supplemental data

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Half map: #1

Fileemd_4082_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_4082_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : 30S-IF1-IF2-IF3-mRNA-tRNA pre-initiation complex (state-II)

EntireName: 30S-IF1-IF2-IF3-mRNA-tRNA pre-initiation complex (state-II)
Components
  • Complex: 30S-IF1-IF2-IF3-mRNA-tRNA pre-initiation complex (state-II)

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Supramolecule #1: 30S-IF1-IF2-IF3-mRNA-tRNA pre-initiation complex (state-II)

SupramoleculeName: 30S-IF1-IF2-IF3-mRNA-tRNA pre-initiation complex (state-II)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#26
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 912.9 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.065 mg/mL
BufferpH: 6.5
Component:
ConcentrationName
10.0 mMMES
5.0 mMMagnessium acetate
50.0 mMPotassium chloride
10.0 mMAmmonium chloride
6.0 mMBeta mercaptoethanol
GridModel: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 90.0 K / Max: 100.0 K
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number grids imaged: 1 / Number real images: 3200 / Average exposure time: 1.5 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 803433
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1hr0

Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 8423
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER / Target criteria: FSC

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