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- EMDB-2239: Cryo-electron microscopy structure of the Trypanosoma brucei 80S ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2239
TitleCryo-electron microscopy structure of the Trypanosoma brucei 80S ribosome
Map dataTrypanosoma Brucei 80S Ribosome cryo-em reconstruction filtered according to the local FSC at 0.5
Sample
  • Sample: Trypanosoma Brucei 80S Ribosome
  • Complex: 80S RibosomeEukaryotic ribosome
KeywordsTrypanosoma / brucei / 80S / ribosome / eukaryotic / kinetoplastids / expansion segments / high-resolution
Function / homology
Function and homology information


organellar small ribosomal subunit / organellar large ribosomal subunit / ciliary transition zone / nuclear lumen / mitochondrial large ribosomal subunit / positive regulation of translational fidelity / phosphate ion binding / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / protein-RNA complex assembly / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) ...organellar small ribosomal subunit / organellar large ribosomal subunit / ciliary transition zone / nuclear lumen / mitochondrial large ribosomal subunit / positive regulation of translational fidelity / phosphate ion binding / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / protein-RNA complex assembly / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rescue of stalled ribosome / maturation of SSU-rRNA / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA / ribosomal large subunit biogenesis / ribosome assembly / small-subunit processome / protein kinase C binding / regulation of cell growth / modification-dependent protein catabolic process / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / protein tag activity / ribosomal small subunit assembly / rRNA processing / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / large ribosomal subunit / ribosome biogenesis / regulation of cell population proliferation / small ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / negative regulation of translation / rRNA binding / protein ubiquitination / ribosome / structural constituent of ribosome / positive regulation of protein phosphorylation / translation / ribonucleoprotein complex / mRNA binding / ubiquitin protein ligase binding / nucleolus / enzyme binding / RNA binding / nucleoplasm / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / Ribosomal protein L30e / Ribosomal protein L28e / Ribosomal protein L23 / Ribosomal L28e/Mak16 / Ribosomal L28e protein family / Ribosomal protein S12e / : / S27a-like superfamily / Ribosomal protein S25 ...: / Ribosomal protein L30e / Ribosomal protein L28e / Ribosomal protein L23 / Ribosomal L28e/Mak16 / Ribosomal L28e protein family / Ribosomal protein S12e / : / S27a-like superfamily / Ribosomal protein S25 / Ribosomal protein S26e signature. / Ribosomal protein S2, eukaryotic / Ribosomal protein S30 / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein L29e / Ribosomal protein S3, eukaryotic/archaeal / S25 ribosomal protein / Ribosomal protein L10e, conserved site / Ribosomal protein S26e / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein L10e / Ribosomal protein S19A/S15e / Ribosomal L29e protein family / Ribosomal protein S30 / Ribosomal protein S12e signature. / Ribosomal protein S17e / Ribosomal protein S17e-like superfamily / Ribosomal protein S27a / Ribosomal protein S2, eukaryotic/archaeal / Ribosomal protein S19e / Ribosomal_S19e / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S8e, conserved site / 40S ribosomal protein S11, N-terminal / 40S ribosomal protein S1/3, eukaryotes / Ribosomal protein S6, eukaryotic / Ribosomal protein S7e / 40S ribosomal protein S4, C-terminal domain / Ribosomal protein L1, conserved site / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal S17 / Ribosomal protein L44e / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal protein L27e / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein L27e superfamily / Ribosomal protein S21e / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal protein S19e / Ribosomal protein S27, zinc-binding domain superfamily / Ribosomal L38e protein family / Ribosomal L22e protein family / 40S Ribosomal protein S10 / 60S ribosomal protein L35 / Ribosomal protein L1 / Ribosomal protein S17, archaeal/eukaryotic / Ribosomal protein S27 / Ribosomal protein S28e conserved site / Ribosomal protein S6/S6e/A/B/2, conserved site / Ribosomal protein S28e / Ribosomal protein L30e, conserved site / 40S ribosomal protein S4 C-terminus / Ribosomal protein S4e, N-terminal / Ribosomal protein S23, eukaryotic/archaeal / Ribosomal_S17 N-terminal / Plectin/S10, N-terminal / Ribosomal protein S3Ae / Ribosomal S3Ae family / Ribosomal protein L44 / Ribosomal protein L34Ae / Ribosomal protein S7e / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Plectin/S10 domain / Ribosomal protein S8e / Ribosomal L27e protein family / Ribosomal protein S4, KOW domain / Ribosomal Protein L6, KOW domain / Ribosomal protein S5/S7, eukaryotic/archaeal / Ribosomal protein S4e / Ribosomal protein S4e, central region / Ribosomal protein S4e, central domain superfamily / Ribosomal protein S6e / Ribosomal protein S13/S15, N-terminal / Ribosomal protein S15P / Ribosomal S13/S15 N-terminal domain / Ribosomal protein S6e / Ribosomal protein S4/S9, eukaryotic/archaeal / Ribosomal protein L30/YlxQ / Ribosomal protein L13e / Ribosomal protein L13e / RS4NT (NUC023) domain / Ribosomal protein L34e / Ribosomal protein L1 signature. / Ribosomal protein L37ae
Similarity search - Domain/homology
60S ribosomal protein L44 / Ubiquitin-ribosomal protein eL40 fusion protein / Large ribosomal subunit protein eL18 / Probable 60S ribosomal protein L14 / 60S ribosomal protein L27, putative / 60S ribosomal protein L18a / 40S ribosomal protein S17, putative / Guanine nucleotide-binding protein subunit beta-like protein / Small ribosomal subunit protein uS2 / Large ribosomal subunit protein eL22 ...60S ribosomal protein L44 / Ubiquitin-ribosomal protein eL40 fusion protein / Large ribosomal subunit protein eL18 / Probable 60S ribosomal protein L14 / 60S ribosomal protein L27, putative / 60S ribosomal protein L18a / 40S ribosomal protein S17, putative / Guanine nucleotide-binding protein subunit beta-like protein / Small ribosomal subunit protein uS2 / Large ribosomal subunit protein eL22 / 60S ribosomal protein L29 / Large ribosomal subunit protein eL34 / 40S ribosomal protein S27, putative / Ribosomal protein / 40S ribosomal protein S26 / 40S ribosomal protein S21, putative / 40S ribosomal protein S5, putative / Large ribosomal subunit protein eL28 / 60S ribosomal protein L17, putative / 40S ribosomal protein S12 / 40S ribosomal protein S4 / Ribosomal protein L37 / 40S ribosomal protein S2, putative / 40S ribosomal protein S3, putative / 60S ribosomal protein L6, putative / 40S ribosomal protein S24 / 60S ribosomal protein L37a, putative / 60S ribosomal protein L24, putative / 40S ribosomal protein S10, putative / 40S ribosomal protein S18, putative / 60S ribosomal protein L30 / Small ribosomal subunit protein eS1 / 60S ribosomal protein L9, putative / 40S ribosomal protein S23, putative / 40S ribosomal proteins S11, putative / 60S ribosomal protein L32, putative / 40S ribosomal protein S6 / Ribosomal protein L36, putative / Ribosomal protein L15 / 60S ribosomal protein L5, putative / 60S ribosomal protein L26, putative / 60S ribosomal subunit protein L31, putative / 60S ribosomal protein L27a / 60S ribosomal protein L23, putative / 60S ribosomal protein L10, putative / 60S ribosomal protein L11, putative / 40S ribosomal protein S7 / 60S ribosomal protein L38, putative / 60S ribosomal protein L35, putative / 60S ribosomal protein L21E, putative / 40S ribosomal protein S14 / 40S ribosomal protein S33, putative / 60S ribosomal protein L39, putative / 60S ribosomal protein L7, putative / 60S ribosomal protein L13 / 60S ribosomal protein L7a / 40S ribosomal protein S9, putative / 40S ribosomal protein S16, putative / 60S ribosomal protein L2, putative / Ubiquitin-ribosomal protein eL40 fusion protein / 40S ribosomal protein S15, putative / 40S ribosomal protein S25 / 60S ribosomal protein L12, putative / 40S ribosomal protein S8 / 60S ribosomal protein L23a / 60S ribosomal protein L19, putative / 40S ribosomal protein S15a, putative / Ribosomal protein L3, mitochondrial, putative / 60S ribosomal protein L4 / Ribosomal protein S19, putative / 60S ribosomal protein L35A, putative / 40S ribosomal protein S30 / 60S ribosomal protein L13a, putative / 40S ribosomal protein S13, putative
Similarity search - Component
Biological speciesTrypanosoma brucei (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.57 Å
AuthorsHashem Y / des Georges A / Fu J / Buss SN / Jossinet F / Jobe A / Zhang Q / Liao HY / Grassucci B / Bajaj C ...Hashem Y / des Georges A / Fu J / Buss SN / Jossinet F / Jobe A / Zhang Q / Liao HY / Grassucci B / Bajaj C / Westhof E / Madison-Antenucci S / Frank J
CitationJournal: Nature / Year: 2013
Title: High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome.
Authors: Yaser Hashem / Amedee des Georges / Jie Fu / Sarah N Buss / Fabrice Jossinet / Amy Jobe / Qin Zhang / Hstau Y Liao / Robert A Grassucci / Chandrajit Bajaj / Eric Westhof / Susan Madison- ...Authors: Yaser Hashem / Amedee des Georges / Jie Fu / Sarah N Buss / Fabrice Jossinet / Amy Jobe / Qin Zhang / Hstau Y Liao / Robert A Grassucci / Chandrajit Bajaj / Eric Westhof / Susan Madison-Antenucci / Joachim Frank /
Abstract: Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and ...Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.
History
DepositionDec 6, 2012-
Header (metadata) releaseDec 26, 2012-
Map releaseFeb 13, 2013-
UpdateFeb 27, 2013-
Current statusFeb 27, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 108000
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 108000
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v8m
  • Surface level: 108000
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2239.jpg

Downloads & links

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Map

FileDownload / File: emd_2239.map.gz / Format: CCP4 / Size: 172.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTrypanosoma Brucei 80S Ribosome cryo-em reconstruction filtered according to the local FSC at 0.5
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 108000.0 / Movie #1: 108000
Minimum - Maximum-202702.703125 - 483783.78125
Average (Standard dev.)7679.428710940000201 (±33729.66015625)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-179-179-179
Dimensions359359359
Spacing359359359
CellA=B=C: 391.31 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z359359359
origin x/y/z0.0000.0000.000
length x/y/z391.310391.310391.310
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-179-179-179
NC/NR/NS359359359
D min/max/mean-202702.703483783.7817679.429

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Supplemental data

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Sample components

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Entire : Trypanosoma Brucei 80S Ribosome

EntireName: Trypanosoma Brucei 80S Ribosome
Components
  • Sample: Trypanosoma Brucei 80S Ribosome
  • Complex: 80S RibosomeEukaryotic ribosome

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Supramolecule #1000: Trypanosoma Brucei 80S Ribosome

SupramoleculeName: Trypanosoma Brucei 80S Ribosome / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1
Molecular weightTheoretical: 3.305 MDa
Method: Molecular weight estimated form the protein and RNA sequence.

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Supramolecule #1: 80S Ribosome

SupramoleculeName: 80S Ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Trypanosoma brucei (eukaryote) / Strain: TREU 667 / Location in cell: Cytoplasm
Molecular weightTheoretical: 3.3 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

Concentration0.105 mg/mL
BufferpH: 7.2
Details: 20 mM Tris pH 7.2, 100mM MgCl2, 500 mM KCl, 5 mM beta-mercaptoethanol
GridDetails: 300 mesh Copper/Molbydenum holey carbon-coated Quantifoil 2/4 grid (Quantifoil Micro Tools GmbH) containing an additional continuous thin layer of carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK IV / Method: Wait 30 sec, Blot 6 seconds, plunge

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJan 1, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Number real images: 1000 / Average electron dose: 25 e/Å2 / Bits/pixel: 32
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase-flip on each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 5.57 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 164000
DetailsData were processed using SPIDER. The particles windows were automatically extracted from 1000 film-recorded micrographs and inspected manually. Standard SPIDER protocols for reference-based reconstruction, except that contrast transfer function (CTF) of the reconstructions was corrected by phase-flipping the particles using the defocus value estimated for each micrograph and a single reconstruction was obtained from the entire dataset using conjugate gradients with regularization (BP CG in SPIDER).
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3u5b
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

PDB-4v8m:
High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome

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Atomic model buiding 2

Initial modelPDB ID:

2xzm
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

PDB-4v8m:
High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome

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Atomic model buiding 3

Initial modelPDB ID:

3izr
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The structure of the 80S from Yeast (3U5B and others) as well as the structure of the 60S from Tetrahymena thermophila (4A17 and others) were used as starting model for the 60S subunit model. The 40S from Tetrahymena thermophila (2XZM and 2XZN) as well as the 80S from Yeast were used as starting model for the 40S subunit model. The 80S model of Triticum aestivum (3IZR and others) was used to fit missing proteins form the two X-ray structures.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

PDB-4v8m:
High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome

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