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- EMDB-3890: State D (Nsa1-TAP Flag-Ytm1) - Visualizing the assembly pathway o... -

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Entry
Database: EMDB / ID: EMD-3890
TitleState D (Nsa1-TAP Flag-Ytm1) - Visualizing the assembly pathway of nucleolar pre-60S ribosomes
Map dataState D (Nsa1-TAP Flag-Ytm1) - Visualizing the assembly pathway of nucleolar pre-60S ribosmes
Sample
  • Complex: Large subunit biogenesis particle purified via Nsa1-TAP and Flag-Ytm1
Function / homology
Function and homology information


25S rRNA (cytosine2870-C5)-methyltransferase / 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase / Noc2p-Noc3p complex / rRNA (guanosine-2'-O-)-methyltransferase activity / snoRNA release from pre-rRNA / protein-RNA complex remodeling / nuclear division / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA (uridine-2'-O-)-methyltransferase activity / rRNA (guanine) methyltransferase activity ...25S rRNA (cytosine2870-C5)-methyltransferase / 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase / Noc2p-Noc3p complex / rRNA (guanosine-2'-O-)-methyltransferase activity / snoRNA release from pre-rRNA / protein-RNA complex remodeling / nuclear division / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA (uridine-2'-O-)-methyltransferase activity / rRNA (guanine) methyltransferase activity / endonucleolytic cleavage in ITS1 upstream of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA (cytosine-C5-)-methyltransferase activity / PeBoW complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / nuclear pre-replicative complex / rRNA base methylation / rRNA primary transcript binding / ATP-dependent activity, acting on RNA / positive regulation of ATP-dependent activity / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA methylation / maturation of 5.8S rRNA / hexon binding / pre-mRNA 5'-splice site binding / preribosome, small subunit precursor / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / proteasome binding / Major pathway of rRNA processing in the nucleolus and cytosol / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / ribosomal large subunit binding / 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 / nuclear-transcribed mRNA catabolic process / ATPase activator activity / L13a-mediated translational silencing of Ceruloplasmin expression / translational elongation / ribosomal large subunit export from nucleus / 90S preribosome / DNA replication initiation / chromosome organization / regulation of translational fidelity / protein-RNA complex assembly / ribonucleoprotein complex binding / ribosomal subunit export from nucleus / maturation of LSU-rRNA / translation initiation factor activity / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / proteasome complex / nuclear periphery / assembly of large subunit precursor of preribosome / ribosomal large subunit biogenesis / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / cytosolic ribosome assembly / small-subunit processome / macroautophagy / maintenance of translational fidelity / protein catabolic process / rRNA processing / ribosomal small subunit biogenesis / protein transport / ribosome biogenesis / viral capsid / nuclear envelope / protein-macromolecule adaptor activity / ATPase binding / 5S rRNA binding / large ribosomal subunit rRNA binding / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / RNA helicase activity / negative regulation of translation / rRNA binding / RNA helicase / ribosome / structural constituent of ribosome / translation / cell division / GTPase activity / mRNA binding / chromatin binding / host cell nucleus / GTP binding / nucleolus / ATP hydrolysis activity / DNA binding / RNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Nucleolar complex-associated protein 3, N-terminal / Nucleolar complex-associated protein 3 / Nucleolar complex-associated protein / WDR74/Nsa1 / WD repeat WDR12/Ytm1 / Ribosome biogenesis factor, NIP7 / RNA (C5-cytosine) methyltransferase, NOP2 / UPF0113, pre-PUA domain / UPF0113 Pre-PUA domain / Ribosomal RNA processing protein 1-like ...Nucleolar complex-associated protein 3, N-terminal / Nucleolar complex-associated protein 3 / Nucleolar complex-associated protein / WDR74/Nsa1 / WD repeat WDR12/Ytm1 / Ribosome biogenesis factor, NIP7 / RNA (C5-cytosine) methyltransferase, NOP2 / UPF0113, pre-PUA domain / UPF0113 Pre-PUA domain / Ribosomal RNA processing protein 1-like / UPF0113 PUA domain / Nucleolar protein,Nop52 / UPF0113, PUA domain / Mak16 protein / Nop2p / Bacterial Fmu (Sun)/eukaryotic nucleolar NOL1/Nop2p, conserved site / Mak16 protein C-terminal region / NOL1/NOP2/sun family signature. / Ribosome biogenesis protein 15, RNA recognition motif / Ribosomal RNA methyltransferase, SPB1-like, C-terminal / Ribosomal RNA methyltransferase Spb1, domain of unknown function DUF3381 / AdoMet-dependent rRNA methyltransferase SPB1-like / Spb1 C-terminal domain / Ribosomal RNA methyltransferase Spb1, DUF3381 / Eukaryotic rRNA processing / Eukaryotic rRNA processing protein EBP2 / : / Ribosomal RNA large subunit methyltransferase E / Ribosome biogenesis protein BRX1 / CCAAT-binding factor / DDX18/Has1, DEAD-box helicase domain / CBF/Mak21 family / Ribosome biogenesis protein Nop16 / Ribosome biogenesis protein Nop16 / Domain of unknown function DUF4217 / Domain of unknown function (DUF4217) / DUF4217 / Domain of unknown function DUF2423 / YBL028C ribosome biogenesis factor, N-terminal domain / BOP1, N-terminal domain / WD repeat BOP1/Erb1 / BOP1NT (NUC169) domain / BOP1NT (NUC169) domain / NLE / NLE (NUC135) domain / SAM-dependent methyltransferase RsmB/NOP2-type / RNA (C5-cytosine) methyltransferase / : / 16S rRNA methyltransferase RsmB/F / SAM-dependent MTase RsmB/NOP-type domain profile. / Putative RNA-binding Domain in PseudoUridine synthase and Archaeosine transglycosylase / PUA domain / Guanine nucleotide-binding protein-like 3, N-terminal domain / GNL3L/Grn1 putative GTPase / U3 snoRNP protein/Ribosome production factor 1 / PUA domain superfamily / PUA domain profile. / : / Pescadillo / Pescadillo N-terminus / GTP-binding protein, orthogonal bundle domain superfamily / Ribosomal biogenesis NSA2 family / Ribosome assembly factor Mrt4 / : / BRCT domain, a BRCA1 C-terminus domain / NOG, C-terminal / Nucleolar GTP-binding protein 1 / NOGCT (NUC087) domain / Nucleolar GTP-binding protein 1, Rossman-fold domain / NOG1, N-terminal helical domain / Nucleolar GTP-binding protein 1 (NOG1) / NOG1 N-terminal helical domain / Brix domain / Brix domain / Brix domain profile. / Brix / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain profile. / OBG-type guanine nucleotide-binding (G) domain / OBG-type guanine nucleotide-binding (G) domain profile. / Translation initiation factor IF6 / eIF-6 family / translation initiation factor 6 / 50S ribosome-binding GTPase / DEAD-box subfamily ATP-dependent helicases signature. / Pre-hexon-linking protein VIII / Adenovirus hexon associated protein, protein VIII / RNA helicase, DEAD-box type, Q motif / ATP-dependent RNA helicase DEAD-box, conserved site / DEAD-box RNA helicase Q motif profile. / GTP binding domain / PUA-like superfamily / 50S ribosomal protein L10, insertion domain superfamily / Ribosomal L28e/Mak16 / Ribosomal L28e protein family / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / metallochaperone-like domain / TRASH domain / breast cancer carboxy-terminal domain
Similarity search - Domain/homology
Large ribosomal subunit protein eL36B / Large ribosomal subunit protein uL23 / 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 eL15A / Large ribosomal subunit protein eL22A / Large ribosomal subunit protein eL27A ...Large ribosomal subunit protein eL36B / Large ribosomal subunit protein uL23 / 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 eL15A / Large ribosomal subunit protein eL22A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein uL14A / Pre-hexon-linking protein VIII / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Large ribosomal subunit protein uL4A / Protein MAK16 / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase / Large ribosomal subunit protein uL13A / Ribosome assembly factor MRT4 / Ribosomal RNA-processing protein 1 / rRNA-processing protein EBP2 / Large ribosomal subunit protein eL14A / Large ribosomal subunit protein eL32 / UPF0642 protein YBL028C / Proteasome-interacting protein CIC1 / Ribosome production factor 1 / Nucleolar protein 16 / Nuclear GTP-binding protein NUG1 / Ribosome biogenesis protein NSA2 / Ribosome biogenesis protein RLP7 / 25S rRNA (cytosine(2870)-C(5))-methyltransferase / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Ribosome biogenesis protein NSA1 / Pescadillo homolog / Ribosome biogenesis protein 15 / Large ribosomal subunit protein eL34A / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Nucleolar GTP-binding protein 1 / ATP-dependent RNA helicase HAS1 / Ribosome biogenesis protein ERB1 / Nucleolar complex-associated protein 3 / Ribosome biogenesis protein RLP24 / Ribosome biogenesis protein BRX1 / 60S ribosome subunit biogenesis protein NIP7 / Ribosome biogenesis protein YTM1 / Eukaryotic translation initiation factor 6 / Large ribosomal subunit protein eL13A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsKater L / Thoms M / Barrio-Garcia C / Cheng J / Ismail S / Ahmed YL / Bange G / Kressler D / Berninghausen O / Sinning I ...Kater L / Thoms M / Barrio-Garcia C / Cheng J / Ismail S / Ahmed YL / Bange G / Kressler D / Berninghausen O / Sinning I / Hurt E / Beckmann R
CitationJournal: Cell / Year: 2017
Title: Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes.
Authors: Lukas Kater / Matthias Thoms / Clara Barrio-Garcia / Jingdong Cheng / Sherif Ismail / Yasar Luqman Ahmed / Gert Bange / Dieter Kressler / Otto Berninghausen / Irmgard Sinning / Ed Hurt / Roland Beckmann /
Abstract: Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural ...Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.
History
DepositionSep 29, 2017-
Header (metadata) releaseNov 15, 2017-
Map releaseDec 27, 2017-
UpdateDec 27, 2017-
Current statusDec 27, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6em5
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6em5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3890.png

Downloads & links

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Map

FileDownload / File: emd_3890.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationState D (Nsa1-TAP Flag-Ytm1) - Visualizing the assembly pathway of nucleolar pre-60S ribosmes
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 420 pix.
= 455.28 Å		1.08 Å/pix.
x 420 pix.
= 455.28 Å		1.08 Å/pix.
x 420 pix.
= 455.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.084 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.041138835 - 0.12395228
Average (Standard dev.)0.00065085466 (±0.007468635)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 455.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0841.0841.084
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z455.280455.280455.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0410.1240.001

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Supplemental data

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Sample components

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Entire : Large subunit biogenesis particle purified via Nsa1-TAP and Flag-Ytm1

EntireName: Large subunit biogenesis particle purified via Nsa1-TAP and Flag-Ytm1
Components
  • Complex: Large subunit biogenesis particle purified via Nsa1-TAP and Flag-Ytm1

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Supramolecule #1: Large subunit biogenesis particle purified via Nsa1-TAP and Flag-Ytm1

SupramoleculeName: Large subunit biogenesis particle purified via Nsa1-TAP and Flag-Ytm1
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 27.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 34453
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-6em5:
State D architectural model (Nsa1-TAP Flag-Ytm1) - Visualizing the assembly pathway of nucleolar pre-60S ribosomes

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