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- EMDB-10027: Mitochondrial 28S ribosome with IF3 (masked refined IF3) -

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Basic information

Entry
Database: EMDB / ID: EMD-10027
TitleMitochondrial 28S ribosome with IF3 (masked refined IF3)
Map dataLocal resolution filtered, masked sharpened map
Sample
  • Complex: IF3 bound mitochondrial translation pre-initiation complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsItoh Y / Khawaja A / Rorbach J / Amunts A
CitationJournal: Nat Commun / Year: 2020
Title: Distinct pre-initiation steps in human mitochondrial translation.
Authors: Anas Khawaja / Yuzuru Itoh / Cristina Remes / Henrik Spåhr / Olessya Yukhnovets / Henning Höfig / Alexey Amunts / Joanna Rorbach /
Abstract: Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two ...Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.
History
DepositionJun 3, 2019-
Header (metadata) releaseJul 1, 2020-
Map releaseJul 1, 2020-
UpdateJan 27, 2021-
Current statusJan 27, 2021Processing 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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10027.png

Downloads & links

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Map

FileDownload / File: emd_10027.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal resolution filtered, masked sharpened map
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.49442405 - 0.6772307
Average (Standard dev.)0.0043452927 (±0.045004737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin229174137
Dimensions114111162
Spacing111114162
CellA: 92.13 Å / B: 94.619995 Å / C: 134.45999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z111114162
origin x/y/z0.0000.0000.000
length x/y/z92.13094.620134.460
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS174229137
NC/NR/NS111114162
D min/max/mean-0.4940.6770.004

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Supplemental data

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Mask #1

Fileemd_10027_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_10027_half_map_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_10027_half_map_2.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : IF3 bound mitochondrial translation pre-initiation complex

EntireName: IF3 bound mitochondrial translation pre-initiation complex
Components
  • Complex: IF3 bound mitochondrial translation pre-initiation complex

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Supramolecule #1: IF3 bound mitochondrial translation pre-initiation complex

SupramoleculeName: IF3 bound mitochondrial translation pre-initiation complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#32
Details: Small subunit of mitochondrial ribosome in complex with mitochondrial IF3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.1 MDa

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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

Concentration0.7 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
0.1 MKClPotassium chloride
25.0 mMHEPES-KOH4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-potassium hydroxide buffer
5.0 mMMg(CH3COO)2Magnesium acetate
0.25 mMC10H17N6O13P35'-Guanylyl imidodiphosphate
0.05 %C24H46O11n-Dodecyl-beta-D-maltopyranoside
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 0.25 µm / Calibrated magnification: 165000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 2-20 / Number grids imaged: 1 / Number real images: 13831 / Average exposure time: 4.0 sec. / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1103314
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: EMDB MAP
EMDB ID:

2880

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.2) / Number images used: 379761
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3j9m

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Cross-correlation coefficient

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