+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6654 | |||||||||
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Title | Negative stain 3DEM structure of Rrp5 | |||||||||
Map data | Reconstruction of Rrp5:Rok1 | |||||||||
Sample |
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Keywords | Rrp5 / 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) / 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) / rRNA primary transcript binding / 90S preribosome assembly / ATP-dependent activity, acting on RNA / poly(U) RNA binding / U3 snoRNA binding / snoRNA binding / Major pathway of rRNA processing in the nucleolus and cytosol / 90S preribosome / 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) / RNA processing / maturation of SSU-rRNA / small-subunit processome / rRNA processing / RNA helicase activity / RNA helicase / mRNA binding / nucleolus / RNA binding / nucleoplasm / ATP binding Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 30.0 Å | |||||||||
Authors | Khoshnevis S / Askenasy I / Johnson MC / Young-Erdos CL / Stroupe ME / Karbstein K | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6654.map.gz | 6.1 MB | EMDB map data format | |
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Header (meta data) | emd-6654-v30.xml emd-6654.xml | 12 KB 12 KB | Display Display | EMDB header |
Images | 400_6654.gif 80_6654.gif | 21.2 KB 5.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6654 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6654 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_6654.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of Rrp5:Rok1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Saccharomyces cerevisiae Rrp5 bound to Rok1
Entire | Name: Saccharomyces cerevisiae Rrp5 bound to Rok1 |
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Components |
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-Supramolecule #1000: Saccharomyces cerevisiae Rrp5 bound to Rok1
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 240 KDa |
-Macromolecule #1: Ribosomal RNA Processing 5
Macromolecule | Name: 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 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: By 4741 / synonym: Yeast / Location in cell: nucleolus |
Molecular weight | Experimental: 200 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta2 (DE3) / Recombinant plasmid: pGEX-6-P3 |
Sequence | UniProtKB: 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 |
-Macromolecule #2: ATP-dependent RNA helicase ROK1
Macromolecule | Name: 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 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: By 4741 / synonym: Yeast / Location in cell: cytoplasmic |
Molecular weight | Theoretical: 40 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta2 (DE3) / Recombinant plasmid: pGEX-6-P3 |
Sequence | UniProtKB: 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 |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 6.7 / Details: 300 mM NaCl, 10 mM MgCl2, 30 mM MES |
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Staining | Type: NEGATIVE / Details: uranyl formate on floated continuous carbon |
Grid | Details: 400 mesh copper grid, glow-discharged in Gatan Solarus apparatus |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 65555 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Date | Mar 23, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1200 / Average electron dose: 20 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final two d classification | Number classes: 50 |
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Final reconstruction | Algorithm: 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 |
Details | Particles were initially manually picked to determine de novo class averages and then automatically picked. Initial model was determined with random conical tilt. |