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- EMDB-10179: mt-SSU late assembly intermediate of wild-type Trypanosoma brucei... -

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Basic information

Entry
Database: EMDB / ID: EMD-10179
Titlemt-SSU late assembly intermediate of wild-type Trypanosoma brucei brucei
Map datamt-SSU late assembly intermediate of wild-type Trypanosoma brucei brucei
Sample
  • Complex: wild-type mt-SSU late assembly intermediate
Biological speciesTrypanosoma brucei brucei (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsSaurer M / Ramrath D / Niemann M / Calderaro S / Prange C / Mattei S / Scaiola A / Leitner A / Bieri P / Horn EK ...Saurer M / Ramrath D / Niemann M / Calderaro S / Prange C / Mattei S / Scaiola A / Leitner A / Bieri P / Horn EK / Leibundgut M / Schneider A / Boehringer D / Ban N
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Science / Year: 2019
Title: Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery.
Authors: Martin Saurer / David J F Ramrath / Moritz Niemann / Salvatore Calderaro / Céline Prange / Simone Mattei / Alain Scaiola / Alexander Leitner / Philipp Bieri / Elke K Horn / Marc Leibundgut ...Authors: Martin Saurer / David J F Ramrath / Moritz Niemann / Salvatore Calderaro / Céline Prange / Simone Mattei / Alain Scaiola / Alexander Leitner / Philipp Bieri / Elke K Horn / Marc Leibundgut / Daniel Boehringer / André Schneider / Nenad Ban /
Abstract: Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome ...Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo-electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.
History
DepositionAug 3, 2019-
Header (metadata) releaseSep 25, 2019-
Map releaseSep 25, 2019-
UpdateSep 25, 2019-
Current statusSep 25, 2019Processing 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 radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10179.png

Downloads & links

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Map

FileDownload / File: emd_10179.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmt-SSU late assembly intermediate of wild-type Trypanosoma brucei brucei
Voxel sizeX=Y=Z: 1.69531 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.07075221 - 0.13252065
Average (Standard dev.)0.0017988548 (±0.00947507)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 433.99936 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.695308593751.695308593751.69530859375
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z433.999433.999433.999
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0710.1330.002

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Supplemental data

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Sample components

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Entire : wild-type mt-SSU late assembly intermediate

EntireName: wild-type mt-SSU late assembly intermediate
Components
  • Complex: wild-type mt-SSU late assembly intermediate

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Supramolecule #1: wild-type mt-SSU late assembly intermediate

SupramoleculeName: wild-type mt-SSU late assembly intermediate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#103
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote) / Strain: Lister strain 427

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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

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsMitochondrial ribosomal small subunit middle assembly intermediate of wild-type Trypanosoma brucei brucei Lister strain 427

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.5 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 129032 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 13503 / Average exposure time: 1.5 sec. / Average electron dose: 75.0 e/Å2
Details: Images were collected in movie-mode at 40 frames per second
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1243432
CTF correctionSoftware - Name: Gctf (ver. 1.06) / Details: On the fly in RELION
Startup modelType of model: OTHER
Details: The startup model was obtained from 3D classification of all particles classified as mt-SSU (mt-SSU initial reference was determined ab-initio using RELION 3D initial model routine based on SGD).
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.5) / Number images used: 6955

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Atomic model buiding 1

DetailsStarting models of ribosomal proteins of the T. brucei mitochondrial ribosomal small subunit were taken from PDB 6HIW. Mitchondrial ribosomal small subunit assembly factors were built de novo.
RefinementProtocol: OTHER

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