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- EMDB-2167: Cryo-EM structure of the 60S-Arx1-Rei1-Jjj1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2167
TitleCryo-EM structure of the 60S-Arx1-Rei1-Jjj1 complex
Map dataCryo-EM reconstruction of the 60S-Arx1-Rei1-Jjj1 complex
Sample
  • Sample: 60S ribosomal subunit in complex with Arx1, Rei1, and Jjj1
  • Complex: 60S ribosomal subunit
  • Protein or peptide: Arx1
  • Protein or peptide: Rei1
  • Protein or peptide: Jjj1
Keywordslarge ribosomal subunit / ribosome biogenesis / ribosome maturation factor
Function / homology: / : / : / DnaJ domain, conserved site / Zinc finger, double-stranded RNA binding / DnaJ domain / Peptidase M24
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.3 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-
UpdateDec 12, 2012-
Current statusDec 12, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 24.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 24.6
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2167.jpg

Downloads & links

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Map

FileDownload / File: emd_2167.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the 60S-Arx1-Rei1-Jjj1 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.17 Å/pix.
x 128 pix.
= 405.76 Å		3.17 Å/pix.
x 128 pix.
= 405.76 Å		3.17 Å/pix.
x 128 pix.
= 405.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.17 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 24.600000000000001 / Movie #1: 24.6
Minimum - Maximum-172.466644290000005 - 300.589538570000002
Average (Standard dev.)-9.7839489 (±23.98919678)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 405.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.173.173.17
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z405.760405.760405.760
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/NS128128128
D min/max/mean-172.467300.590-9.784

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Supplemental data

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Sample components

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Entire : 60S ribosomal subunit in complex with Arx1, Rei1, and Jjj1

EntireName: 60S ribosomal subunit in complex with Arx1, Rei1, and Jjj1
Components
  • Sample: 60S ribosomal subunit in complex with Arx1, Rei1, and Jjj1
  • Complex: 60S ribosomal subunit
  • Protein or peptide: Arx1
  • Protein or peptide: Rei1
  • Protein or peptide: Jjj1

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Supramolecule #1000: 60S ribosomal subunit in complex with Arx1, Rei1, and Jjj1

SupramoleculeName: 60S ribosomal subunit in complex with Arx1, Rei1, and Jjj1
type: sample / ID: 1000
Oligomeric state: One monomer each of 60S, Arx1, Rei1, and Jjj1
Number unique components: 4
Molecular weightTheoretical: 2.4 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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Macromolecule #3: Jjj1

MacromoleculeName: Jjj1 / type: protein_or_peptide / ID: 3 / Details: C-terminal His-tag, N-terminal STREP 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: 69 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pASK-IBA45+
SequenceInterPro: DnaJ domain, DnaJ domain, conserved site, INTERPRO: IPR003095, INTERPRO: IPR007087, INTERPRO: IPR015880, Zinc finger, double-stranded RNA binding

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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: 7.6
Details: 20 mM Hepes-NaOH pH 7.6, 100 mM NaCl, 5 mM MgCl2, 5 mM beta-mercaptoethanol, 0.1 mM ZnCl2
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
DateMay 3, 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
DateMay 4, 2011
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: 16.3 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 0.2.
Number images used: 8881

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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

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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

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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

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