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- EMDB-6654: Negative stain 3DEM structure of Rrp5 -

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Basic information

Entry
Database: EMDB / ID: EMD-6654
TitleNegative stain 3DEM structure of Rrp5
Map dataReconstruction of Rrp5:Rok1
Sample
  • Sample: Saccharomyces cerevisiae Rrp5 bound to Rok1
  • Protein or peptide: Ribosomal RNA Processing 5
  • Protein or peptide: ATP-dependent RNA helicase ROK1
KeywordsRrp5 / RNA binding protein / ribosome assembly factor / 40S ribosome / 60S ribosome / Rok1 / DEAD-box protein
Function / homology
Function and homology information


intracellular anatomical structure / snoRNA localization / protein binding / box H/ACA snoRNA binding / endonucleolytic cleavage in ITS1 upstream of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / box C/D sno(s)RNA binding / endonucleolytic cleavage in 5'-ETS of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / endonucleolytic cleavage to generate mature 5'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ATP-dependent activity, acting on RNA / rRNA primary transcript binding ...intracellular anatomical structure / snoRNA localization / protein binding / box H/ACA snoRNA binding / endonucleolytic cleavage in ITS1 upstream of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / box C/D sno(s)RNA binding / endonucleolytic cleavage in 5'-ETS of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / endonucleolytic cleavage to generate mature 5'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ATP-dependent activity, acting on RNA / rRNA primary transcript binding / 90S preribosome assembly / U3 snoRNA binding / poly(U) RNA binding / snoRNA binding / Major pathway of rRNA processing in the nucleolus and cytosol / 90S preribosome / RNA processing / 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) / maturation of SSU-rRNA / small-subunit processome / rRNA processing / RNA helicase activity / RNA helicase / mRNA binding / nucleolus / ATP hydrolysis activity / RNA binding / nucleoplasm / ATP binding
Similarity search - Function
DDX52/Rok1, DEAD-box helicase domain / rRNA biogenesis protein Rrp5 / : / : / : / PRP39 N-terminal HAT repeat / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / DEAD-box subfamily ATP-dependent helicases signature. ...DDX52/Rok1, DEAD-box helicase domain / rRNA biogenesis protein Rrp5 / : / : / : / PRP39 N-terminal HAT repeat / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / DEAD-box subfamily ATP-dependent helicases signature. / ATP-dependent RNA helicase DEAD-box, conserved site / RNA helicase, DEAD-box type, Q motif / DEAD-box RNA helicase Q motif profile. / S1 domain profile. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / S1 domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / Tetratricopeptide-like helical domain superfamily / Tetratricopeptide-like helical domain superfamily / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Nucleic acid-binding, OB-fold / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-dependent RNA helicase ROK1 / rRNA biogenesis protein RRP5
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsKhoshnevis S / Askenasy I / Johnson MC / Young-Erdos CL / Stroupe ME / Karbstein K
CitationJournal: PLoS Biol / Year: 2016
Title: The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.
Authors: Sohail Khoshnevis / Isabel Askenasy / Matthew C Johnson / Maria D Dattolo / Crystal L Young-Erdos / M Elizabeth Stroupe / Katrin Karbstein /
Abstract: DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and ...DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.
History
DepositionMay 12, 2016-
Header (metadata) releaseJun 1, 2016-
Map releaseJun 1, 2016-
UpdateJun 22, 2016-
Current statusJun 22, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.48
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.48
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6654.gif

Downloads & links

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Map

FileDownload / File: emd_6654.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Rrp5:Rok1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 128 pix.
= 358.4 Å		2.8 Å/pix.
x 128 pix.
= 358.4 Å		2.8 Å/pix.
x 128 pix.
= 358.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.48 / Movie #1: 4.48
Minimum - Maximum-4.9492197 - 12.50788307
Average (Standard dev.)0.0 (±0.82991219)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-23-23-23
Dimensions128128128
Spacing128128128
CellA=B=C: 358.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS-23-23-23
NC/NR/NS128128128
D min/max/mean-4.94912.5080.000

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae Rrp5 bound to Rok1

EntireName: Saccharomyces cerevisiae Rrp5 bound to Rok1
Components
  • Sample: Saccharomyces cerevisiae Rrp5 bound to Rok1
  • Protein or peptide: Ribosomal RNA Processing 5
  • Protein or peptide: ATP-dependent RNA helicase ROK1

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Supramolecule #1000: Saccharomyces cerevisiae Rrp5 bound to Rok1

SupramoleculeName: Saccharomyces cerevisiae Rrp5 bound to Rok1 / type: sample / ID: 1000
Details: At a 5-sigma contour, the mass is about 0.25 MDa, which corresponds to about 0.2 MDa from Rrp5 and 0.04 MDa from RNA.
Oligomeric state: 1 / Number unique components: 2
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: Ribosomal RNA Processing 5

MacromoleculeName: Ribosomal RNA Processing 5 / type: protein_or_peptide / ID: 1
Name.synonym: Rrp5, U3 small nucleolar RNA-associated protein RRP5
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: By 4741 / synonym: Yeast / Location in cell: nucleolus
Molecular weightExperimental: 200 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta2 (DE3) / Recombinant plasmid: pGEX-6-P3
SequenceUniProtKB: rRNA biogenesis protein RRP5
GO: RNA processing, RNA binding, protein binding, intracellular anatomical structure
InterPro: Nucleic acid-binding, OB-fold, S1 domain, Tetratricopeptide-like helical domain superfamily, HAT (Half-A-TPR) repeat

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Macromolecule #2: ATP-dependent RNA helicase ROK1

MacromoleculeName: ATP-dependent RNA helicase ROK1 / type: protein_or_peptide / ID: 2 / Name.synonym: Rescuer of KEM1 protein 1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: By 4741 / synonym: Yeast / Location in cell: cytoplasmic
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta2 (DE3) / Recombinant plasmid: pGEX-6-P3
SequenceUniProtKB: ATP-dependent RNA helicase ROK1
GO: 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)
InterPro: DEAD/DEAH box helicase domain

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.7 / Details: 300 mM NaCl, 10 mM MgCl2, 30 mM MES
StainingType: NEGATIVE / Details: uranyl formate on floated continuous carbon
GridDetails: 400 mesh copper grid, glow-discharged in Gatan Solarus apparatus
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateMar 23, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1200 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 65555 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsParticles were initially manually picked to determine de novo class averages and then automatically picked. Initial model was determined with random conical tilt.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: EMAN
Details: Final maps were calculated from two averaged datasets.
Number images used: 8500
Final two d classificationNumber classes: 50

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