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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: 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
SampleLarge subunit biogenesis particle purified via Nsa1-TAP and Flag-Ytm1
Function/homology27S pre-rRNA (guanosine2922-2'-O)-methyltransferase / Nucleolar complex-associated protein 3 / Noc2p-Noc3p complex / AdoMet-dependent rRNA methyltransferase, Spb1 / ec:2.1.1.310: / RNA (C5-cytosine) methyltransferase, NOP2 / Ribosomal RNA methyltransferase Spb1, domain of unknown function DUF3381 / Nucleolar complex-associated protein 3, N-terminal / Ribosomal RNA methyltransferase, Spb1, C-terminal / Ribosome biogenesis factor, NIP7 ...27S pre-rRNA (guanosine2922-2'-O)-methyltransferase / Nucleolar complex-associated protein 3 / Noc2p-Noc3p complex / AdoMet-dependent rRNA methyltransferase, Spb1 / ec:2.1.1.310: / RNA (C5-cytosine) methyltransferase, NOP2 / Ribosomal RNA methyltransferase Spb1, domain of unknown function DUF3381 / Nucleolar complex-associated protein 3, N-terminal / Ribosomal RNA methyltransferase, Spb1, C-terminal / Ribosome biogenesis factor, NIP7 / rRNA (guanine) methyltransferase activity / Domain of unknown function (DUF3381) / Spb1 C-terminal domain / Nucleolar complex-associated protein / CCAAT-binding factor / Ribosome biogenesis factor NIP7-like / WD repeat WDR12/Ytm1 / Ribosomal RNA large subunit methyltransferase E / CBF/Mak21 family / rRNA (cytosine-C5-)-methyltransferase activity / rRNA (uridine-2'-O-)-methyltransferase activity / Guanine nucleotide-binding protein-like 3, N-terminal domain / Nop2p / Uncharacterised protein family UPF0642 / Ribosomal RNA small subunit methyltransferase F, N-terminal / Uncharacterised protein family (UPF0113) / Eukaryotic rRNA processing / GTP-binding protein, orthogonal bundle domain superfamily / Mak16 protein / Bacterial Fmu (Sun)/eukaryotic nucleolar NOL1/Nop2p, conserved site / cell cycle DNA replication / NOL1/NOP2/sun family signature. / snoRNA release from pre-rRNA / Ribosome biogenesis protein Nop16 / maturation of 5.8S rRNA / Ribosome biogenesis protein BRX1 / GNL3L/Grn1 putative GTPase / Protein of unknown function (DUF2423) / WDR74/Nsa1 / Ribosome assembly factor Mrt4 / Nucleolar, Nop52 / WD repeat BOP1/Erb1 / BOP1, N-terminal domain / Nucleolar GTP-binding protein 1 / NOG, C-terminal / Nucleolar GTP-binding protein 1, Rossman-fold domain / Domain of unknown function DUF4217 / nuclear division / NLE / Pescadillo / Eukaryotic rRNA processing protein EBP2 / Ribosome biogenesis protein 15, RNA recognition motif / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Mak16 protein C-terminal region / Ribosome biogenesis protein Nop16 / regulation of rRNA processing / N-terminal domain of 16S rRNA methyltransferase RsmF / RNA (C5-cytosine) methyltransferase / BOP1NT (NUC169) domain / SAM-dependent methyltransferase RsmB/NOP2-type / SAM-dependent MTase RsmB/NOP-type domain profile. / Domain of unknown function (DUF4217) / Nucleolar protein,Nop52 / PeBoW complex / NOGCT (NUC087) domain / Nucleolar GTP-binding protein 1 (NOG1) / Pescadillo N-terminus / rRNA primary transcript binding / NLE (NUC135) domain / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain profile. / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain / Brix domain / Brix domain profile. / Translation initiation factor IF6 / OBG-type guanine nucleotide-binding (G) domain / OBG-type guanine nucleotide-binding (G) domain profile. / 16S rRNA methyltransferase RsmB/F / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / PUA domain superfamily / nuclear pre-replicative complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / actomyosin contractile ring assembly / PUA domain / PUA domain profile. / eIF-6 family / Brix domain / mature ribosome assembly / regulation of cell division / rRNA base methylation / RNA-dependent ATPase activity / ribosomal subunit export from nucleus / Ribosomal L28e/Mak16 / chromosome organization / BRCT domain, a BRCA1 C-terminus domain / nuclear-transcribed mRNA catabolic process / 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) / GTP binding domain / Ribosomal L28e protein family / ATPase activator activity
Function and homology information
SourceSaccharomyces cerevisiae / / yeast /
MethodCryo EM / single particle reconstruction / 4.2 Å resolution
AuthorsKater 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.
Copyright: 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Validation ReportPDB-ID: 6em5

SummaryFull reportAbout validation report
DateDeposition: Sep 29, 2017 / Header (metadata) release: Nov 15, 2017 / Map release: Dec 27, 2017 / Last update: Dec 27, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • 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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3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_3890.map.gz (map file in CCP4 format, 296353 KB)
Projections & slices

Image control

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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.084 Å
Density
Contour Level:0.025 (by author), 0.025 (movie #1):
Minimum - Maximum-0.041138835 - 0.12395228
Average (Standard dev.)0.00065085466 (0.007468635)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions420420420
Origin000
Limit419419419
Spacing420420420
CellA=B=C: 455.28 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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
Number of components: 1

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Component #1: protein, Large subunit biogenesis particle purified via Nsa1-TAP ...

ProteinName: Large subunit biogenesis particle purified via Nsa1-TAP and Flag-Ytm1
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae / / yeast /

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: Cryo EM
Sample solutionpH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 27 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON II (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 34453
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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Atomic model buiding

Output model

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