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- EMDB-2169: Cryo-EM structure of the 60S-Arx1-Rei1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2169
TitleCryo-EM structure of the 60S-Arx1-Rei1 complex
Map dataCryo-EM reconstruction of the 60S-Arx1-Rei1 complex
Sample
  • Sample: 60S ribosomal subunit in complex with Arx1 and Rei1
  • Complex: 60S ribosomal subunit
  • Protein or peptide: Arx1
  • Protein or peptide: Rei1
Keywordslarge ribosomal subunit / ribosome biogenesis / ribosome maturation factor
Function / homology
Function and homology information


Hydrolases / hexon binding / pre-mRNA 5'-splice site binding / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / response to cycloheximide / protein kinase activator activity / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) ...Hydrolases / hexon binding / pre-mRNA 5'-splice site binding / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / response to cycloheximide / protein kinase activator activity / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / negative regulation of mRNA splicing, via spliceosome / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / translational elongation / ribosomal large subunit export from nucleus / 90S preribosome / regulation of translational fidelity / protein-RNA complex assembly / ribonucleoprotein complex binding / translational termination / maturation of LSU-rRNA / Neutrophil degranulation / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ribosomal large subunit biogenesis / translational initiation / macroautophagy / maintenance of translational fidelity / modification-dependent protein catabolic process / protein tag activity / rRNA processing / metallopeptidase activity / ribosome biogenesis / viral capsid / 5S rRNA binding / large ribosomal subunit rRNA binding / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / negative regulation of translation / rRNA binding / ribosome / protein ubiquitination / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / ubiquitin protein ligase binding / host cell nucleus / nucleolus / proteolysis / RNA binding / nucleoplasm / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
: / : / Ribosomal protein L12/P1/P2 family / Ribosomal protein P1/P2, N-terminal domain / : / 60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / Pre-hexon-linking protein VIII / Adenovirus hexon associated protein, protein VIII ...: / : / Ribosomal protein L12/P1/P2 family / Ribosomal protein P1/P2, N-terminal domain / : / 60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / Pre-hexon-linking protein VIII / Adenovirus hexon associated protein, protein VIII / Peptidase M24 / Creatinase/aminopeptidase-like / 50S ribosomal protein L10, insertion domain superfamily / : / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein L29e / Ribosomal L29e protein family / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal L38e protein family / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein L44e signature. / Ribosomal protein L10e, conserved site / Ribosomal protein L10e signature. / Ribosomal protein L10e / Ribosomal protein L13e / Ribosomal protein L13e / Ribosomal protein L19, eukaryotic / 60S ribosomal protein L18a/ L20, eukaryotes / : / Ribosomal protein L24e, conserved site / Ribosomal protein L19/L19e conserved site / Ribosomal protein L19e signature. / Ribosomal protein L24e signature. / Ribosomal protein L44e / Ribosomal protein L44 / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / : / Ribosomal protein L30e signature 1. / Ribosomal protein L6e signature. / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / Ribosomal protein 50S-L18Ae/60S-L20/60S-L18A / Ribosomal L40e family / Ribosomal proteins 50S-L18Ae/60S-L20/60S-L18A / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / Ribosomal protein L23/L25, N-terminal / Ribosomal protein L23, N-terminal domain / Ribosomal protein L30e signature 2. / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein L30e, conserved site / Ribosomal protein L27e / Ribosomal protein L27e superfamily / Ribosomal L27e protein family / Ribosomal protein L36e signature. / Ribosomal protein L39e, conserved site / Ribosomal protein L39e signature. / Ribosomal protein L34Ae / Ribosomal protein L34e / Ribosomal protein L14e domain / Ribosomal protein L35Ae, conserved site / 60S ribosomal protein L19 / Ribosomal protein L14 / Ribosomal protein L35Ae signature. / Ribosomal protein L30/YlxQ / Ribosomal Protein L6, KOW domain / Ribosomal protein L7A/L8 / Ribosomal protein L13, eukaryotic/archaeal / 60S ribosomal protein L35 / Ribosomal protein L6e / 60S ribosomal protein L6E / Ribosomal protein L14 / Ribosomal protein L14, KOW motif / Ribosomal protein L37ae / Ribosomal L37ae protein family / 60S ribosomal protein L4, C-terminal domain / 60S ribosomal protein L4 C-terminal domain / Ribosomal protein L7, eukaryotic / Ribosomal protein L31e, conserved site / Ribosomal protein L31e signature. / Ribosomal_L19e / Ribosomal protein L30, N-terminal / Ribosomal L30 N-terminal domain / Ribosomal protein L19/L19e / Ribosomal protein L19/L19e, domain 1 / Ribosomal protein L19/L19e superfamily / Ribosomal protein L19e
Similarity search - Domain/homology
Large ribosomal subunit protein uL15 / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein P1A / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A ...Large ribosomal subunit protein uL15 / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein P1A / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL29 / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL22A / Large ribosomal subunit protein uL5A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Ubiquitin-ribosomal protein eL40A fusion protein / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein eL42A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL2A / Pre-hexon-linking protein VIII / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Small ribosomal subunit protein eS32A / Large ribosomal subunit protein uL4A / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL13A / Large ribosomal subunit protein eL14A / Large ribosomal subunit protein eL32 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Large ribosomal subunit protein eL34A / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Probable metalloprotease ARX1 / Large ribosomal subunit protein eL13A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.1 Å
AuthorsGreber BJ / Boehringer D / Montellese C / Ban N
CitationJournal: Nat Struct Mol Biol / Year: 2012
Title: Cryo-EM structures of Arx1 and maturation factors Rei1 and Jjj1 bound to the 60S ribosomal subunit.
Authors: Basil J Greber / Daniel Boehringer / Christian Montellese / Nenad Ban /
Abstract: Eukaryotic ribosome biogenesis requires many protein factors that facilitate the assembly, nuclear export and final maturation of 40S and 60S particles. We have biochemically characterized ribosomal ...Eukaryotic ribosome biogenesis requires many protein factors that facilitate the assembly, nuclear export and final maturation of 40S and 60S particles. We have biochemically characterized ribosomal complexes of the yeast 60S-biogenesis factor Arx1 and late-maturation factors Rei1 and Jjj1 and determined their cryo-EM structures. Arx1 was visualized bound to the 60S subunit together with Rei1, at 8.1-Å resolution, to reveal the molecular details of Arx1 binding whereby Arx1 arrests the eukaryotic-specific rRNA expansion segment 27 near the polypeptide tunnel exit. Rei1 and Jjj1, which have been implicated in Arx1 recycling, bind in the vicinity of Arx1 and form a network of interactions. We suggest that, in addition to the role of Arx1 during pre-60S nuclear export, the binding of Arx1 conformationally locks the pre-60S subunit and inhibits the premature association of nascent chain-processing factors to the polypeptide tunnel exit.
History
DepositionAug 7, 2012-
Header (metadata) releaseAug 29, 2012-
Map releaseOct 31, 2012-
UpdateSep 10, 2014-
Current statusSep 10, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 35
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 35
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-4v8t
  • Surface level: 35
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2169.jpg

Downloads & links

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Map

FileDownload / File: emd_2169.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the 60S-Arx1-Rei1 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.81 Å/pix.
x 224 pix.
= 405.44 Å		1.81 Å/pix.
x 224 pix.
= 405.44 Å		1.81 Å/pix.
x 224 pix.
= 405.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 35.0 / Movie #1: 35
Minimum - Maximum-221.731964110000007 - 425.313201900000024
Average (Standard dev.)-13.189755440000001 (±29.80069542)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 405.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.811.811.81
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z405.440405.440405.440
α/β/γ90.00090.00090.000
start NX/NY/NZ-32-32-32
NX/NY/NZ646464
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-221.732425.313-13.190

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Supplemental data

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Sample components

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Entire : 60S ribosomal subunit in complex with Arx1 and Rei1

EntireName: 60S ribosomal subunit in complex with Arx1 and Rei1
Components
  • Sample: 60S ribosomal subunit in complex with Arx1 and Rei1
  • Complex: 60S ribosomal subunit
  • Protein or peptide: Arx1
  • Protein or peptide: Rei1

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Supramolecule #1000: 60S ribosomal subunit in complex with Arx1 and Rei1

SupramoleculeName: 60S ribosomal subunit in complex with Arx1 and Rei1 / type: sample / ID: 1000 / Oligomeric state: One monomer each of 60S, Arx1, and Rei1 / Number unique components: 3
Molecular weightTheoretical: 2.3 MDa

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Supramolecule #1: 60S ribosomal subunit

SupramoleculeName: 60S ribosomal subunit / type: complex / ID: 1 / Name.synonym: 60S / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: LSU 60S
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 2.2 MDa

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Macromolecule #1: Arx1

MacromoleculeName: Arx1 / type: protein_or_peptide / ID: 1 / Details: N-terminal His-tag / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 65 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pProEX-htb
SequenceInterPro: Peptidase M24

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Macromolecule #2: Rei1

MacromoleculeName: Rei1 / type: protein_or_peptide / ID: 2 / Details: C-terminal His-tag / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 46 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET24a
SequenceInterPro: INTERPRO: IPR007087, INTERPRO: IPR015880

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Details: 20 mM Hepes-NaOH pH 8.0, 50 mM NaCl, 5 mM MgCl2, 5 mM beta-mercaptoethanol
StainingType: NEGATIVE / Details: cryo
GridDetails: Quantifoil holey carbon grid R2/1
VitrificationCryogen name: ETHANE / Chamber temperature: 80 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: manual plunger / Method: manual blotting

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TECNAI 20
TemperatureAverage: 87 K
DateFeb 21, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Average electron dose: 20 e/Å2
Details: images were acquired using a 2 x 2 frame spot scan (per hole) using a serial EM script
Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 83000
Sample stageSpecimen holder: eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI TECNAI 20
TemperatureAverage: 87 K
DateMar 6, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Average electron dose: 20 e/Å2
Details: images were acquired using a 2 x 2 frame spot scan (per hole) using a serial EM script
Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 83000
Sample stageSpecimen holder: eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Electron microscopy #3

Microscopy ID3
MicroscopeFEI TECNAI 20
TemperatureAverage: 87 K
DateMar 20, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Average electron dose: 20 e/Å2
Details: images were acquired using a 2 x 2 frame spot scan (per hole) using a serial EM script
Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 83000
Sample stageSpecimen holder: eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: per frame
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Fourier amplitudes of the reconstruction were enhanced using the SAXS curve from ribosomes; subsequently, the map was filtered in SPIDER using a butterworth low-pass filter with a filter midpoint of 0.23.
Number images used: 84113

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Atomic model buiding 1

Initial modelPDB ID:

3u5h
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation
Output model

PDB-4v8t:
Cryo-EM Structure of the 60S Ribosomal Subunit in Complex with Arx1 and Rei1

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Atomic model buiding 2

Initial modelPDB ID:

3u5i
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation
Output model

PDB-4v8t:
Cryo-EM Structure of the 60S Ribosomal Subunit in Complex with Arx1 and Rei1

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Atomic model buiding 3

Initial modelPDB ID:

2q8k

SoftwareName: Chimera
DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation
Output model

PDB-4v8t:
Cryo-EM Structure of the 60S Ribosomal Subunit in Complex with Arx1 and Rei1

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