EMDB-4794: Yeast pre-40S Rps20 DeltaLoop C1-Headonly

Basic information

• Entry: Database: EMDB / ID: EMD-4794
• Title: Yeast pre-40S Rps20 DeltaLoop C1-Headonly
• Map data: C1-headonly S20Delta loop yeast pre-40S particle Structural state 1 3D reconstruction of the head only(signal subtraction of the body) Local sharpening with LocScale

Sample

• Complex: C1-S20DeltaLoop State 1 of yeast pre-40S particles purified with Tsr1-TAP as bait, in mutant conditions (deletion of aa 68-78 of Rps20) 3D reconstruction of the pre-40S head only (signal subtraction of the body domain)

Function / homology

Function:

positive regulation of RNA import into nucleus / 18S rRNA (adenine1779-N6/adenine1780-N6)-dimethyltransferase / 18S rRNA (adenine(1779)-N(6)/adenine(1780)-N(6))-dimethyltransferase activity / rRNA modification / rRNA (adenine-N6,N6-)-dimethyltransferase activity / endonucleolytic cleavage 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) / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, LSU-rRNA,5S) / negative regulation of glucose mediated signaling pathway / negative regulation of translational frameshifting / Negative regulators of DDX58/IFIH1 signaling / rRNA primary transcript binding / RMTs methylate histone arginines / positive regulation of translational fidelity / Protein methylation / mTORC1-mediated signalling / Protein hydroxylation / ribosome-associated ubiquitin-dependent protein catabolic process / rRNA methylation / U3 snoRNA binding / GDP-dissociation inhibitor activity / nonfunctional rRNA decay / positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / poly(A)+ mRNA export from nucleus / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / preribosome, small subunit precursor / Ribosomal scanning and start codon recognition / snoRNA binding / Major pathway of rRNA processing in the nucleolus and cytosol / mRNA destabilization / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / 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 / regulation of amino acid metabolic process / L13a-mediated translational silencing of Ceruloplasmin expression / proteasome assembly / 90S preribosome / G-protein alpha-subunit binding / positive regulation of protein kinase activity / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of translational fidelity / Ub-specific processing proteases / ribonucleoprotein complex binding / ribosomal subunit export from nucleus / ribosomal small subunit export from nucleus / translation regulator activity / 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) / DNA-(apurinic or apyrimidinic site) endonuclease activity / cellular response to amino acid starvation / rescue of stalled ribosome / ribosome assembly / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / small-subunit processome / translational initiation / protein kinase C binding / maintenance of translational fidelity / modification-dependent protein catabolic process / cytoplasmic stress granule / protein tag activity / rRNA processing / ribosomal small subunit biogenesis / unfolded protein binding / small ribosomal subunit rRNA binding / ribosome biogenesis / ribosome binding / ribosomal small subunit assembly / small ribosomal subunit / cytosolic small ribosomal subunit / cytoplasmic translation / negative regulation of translation / non-specific serine/threonine protein kinase / protein kinase activity / rRNA binding / ribosome / protein ubiquitination / structural constituent of ribosome / positive regulation of protein phosphorylation / G protein-coupled receptor signaling pathway / translation / negative regulation of gene expression / protein serine kinase activity / protein serine/threonine kinase activity / GTPase activity / mRNA binding / ubiquitin protein ligase binding / GTP binding / nucleolus / mitochondrion / RNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm

Domain/homology:

Serine/threonine-protein kinase Rio2 / RIO2 kinase winged helix domain, N-terminal / Rio2, N-terminal / Tsr1, G-like domain / RIO kinase / RIO-like kinase / RIO1 family / Krr1, KH1 domain / Krr1 KH1 domain / Bystin / Bystin / Ribosomal RNA adenine dimethylase / Ribosomal RNA adenine methylase transferase, conserved site / Ribosomal RNA adenine methylase transferase, N-terminal / Ribosomal RNA adenine dimethylases signature. / Ribosomal RNA adenine dimethylases / Eukaryotic type KH-domain (KH-domain type I) (327.11.2) / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / Ribosome biogenesis protein BMS1/TSR1, C-terminal / AARP2CN / Bms1/Tsr1-type G domain / Ribosome biogenesis protein Bms1/Tsr1 / 40S ribosome biogenesis protein Tsr1 and BMS1 C-terminal / AARP2CN (NUC121) domain / Bms1-type guanine nucleotide-binding (G) domain profile. / AARP2CN (NUC121) domain / Protein of unknown function (DUF663) / K Homology domain, type 1 superfamily / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / : / Ribosomal protein S12e signature. / Ribosomal protein S12e / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal protein S2, eukaryotic / Small (40S) ribosomal subunit Asc1/RACK1 / S27a-like superfamily / 40S Ribosomal protein S10 / Ribosomal protein S10, eukaryotic/archaeal / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S2, eukaryotic/archaeal / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein S17e, conserved site / Ribosomal protein S17e signature. / Ribosomal protein S30 / : / Ribosomal protein S30 / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Ribosomal protein S7e signature. / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein S19e / Ribosomal protein S3Ae, conserved site / Ribosomal protein S19e / Ribosomal protein S3Ae signature. / Ribosomal_S19e / Ribosomal protein S27e signature. / Ribosomal protein S4e, N-terminal, conserved site / Ribosomal protein S4e signature. / 40S ribosomal protein S4, C-terminal domain / 40S ribosomal protein S4 C-terminus / Ribosomal protein S8e, conserved site / Ribosomal protein S8e signature. / Ribosomal protein S19A/S15e / Ribosomal protein S17e / Ribosomal protein S17e-like superfamily / Ribosomal S17 / : / Ribosomal protein S6, eukaryotic / Ribosomal S24e conserved site / Ribosomal protein S24e signature. / 40S ribosomal protein S1/3, eukaryotes / Ribosomal protein S4e, N-terminal / RS4NT (NUC023) domain / 40S ribosomal protein S11, N-terminal / Ribosomal_S17 N-terminal / Ribosomal protein S7e / Ribosomal protein S7e / Ribosomal protein S4, KOW domain / Ribosomal protein S4e / Ribosomal protein S4e, central region / Ribosomal protein S4e, central domain superfamily / Ribosomal family S4e / Ribosomal protein S6/S6e/A/B/2, conserved site / Ribosomal protein S6e signature. / Ribosomal protein S23, eukaryotic/archaeal / Ribosomal protein S24e / Ribosomal protein S24e

Component:

Small ribosomal subunit protein uS4A / Small ribosomal subunit protein eS17A / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS15 / Ubiquitin-ribosomal protein eS31 fusion protein / Small ribosomal subunit protein uS11A / Small ribosomal subunit protein eS19A / Small ribosomal subunit protein eS21A / Small ribosomal subunit protein uS8A / Small ribosomal subunit protein uS12A / Small ribosomal subunit protein eS24A / Small ribosomal subunit protein eS30A / Small ribosomal subunit protein eS4A / Small ribosomal subunit protein eS6A / Small ribosomal subunit protein eS8A / Small ribosomal subunit protein uS17A / Small ribosomal subunit protein uS9A / Small ribosomal subunit protein uS13A / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein eS7A / Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS1A / Small ribosomal subunit protein eS27A / Small ribosomal subunit protein RACK1 / Essential nuclear protein 1 / Small ribosomal subunit protein uS10 / Serine/threonine-protein kinase RIO2 / Small ribosomal subunit protein uS14A / Dimethyladenosine transferase / Small ribosomal subunit protein eS12 / Small ribosomal subunit protein uS19 / Ribosome biogenesis protein TSR1 / Small ribosomal subunit protein eS10A / Small ribosomal subunit protein eS25A / Small ribosomal subunit protein eS28A / Pre-rRNA-processing protein PNO1

• Biological species: Saccharomyces cerevisiae S288c (yeast)
• Method: single particle reconstruction / cryo EM / Resolution: 3.8 Å
• Authors: Shayan R / Mitterer V / Ferreira-Cerca S / Murat G / Rinaldi D / Enne T / Weigl S / Omanic H / Gleizes PE / Kressler D / Pertschy B / Plisson-Chastang C

Funding support

France, 1 items

Organization	Grant number	Country
French National Research Agency	RIBOMAN	France

• Citation: Journal: Nat Commun / Year: 2019; Title: Conformational proofreading of distant 40S ribosomal subunit maturation events by a long-range communication mechanism.; Authors: Valentin Mitterer / Ramtin Shayan / Sébastien Ferreira-Cerca / Guillaume Murat / Tanja Enne / Dana Rinaldi / Sarah Weigl / Hajrija Omanic / Pierre-Emmanuel Gleizes / Dieter Kressler / Celia Plisson-Chastang / Brigitte Pertschy / ; Abstract: Eukaryotic ribosomes are synthesized in a hierarchical process driven by a plethora of assembly factors, but how maturation events at physically distant sites on pre-ribosomes are coordinated is poorly understood. Using functional analyses and cryo-EM, we show that ribosomal protein Rps20 orchestrates communication between two multi-step maturation events across the pre-40S subunit. Our study reveals that during pre-40S maturation, formation of essential contacts between Rps20 and Rps3 permits assembly factor Ltv1 to recruit the Hrr25 kinase, thereby promoting Ltv1 phosphorylation. In parallel, a deeply buried Rps20 loop reaches to the opposite pre-40S side, where it stimulates Rio2 ATPase activity. Both cascades converge to the final maturation steps releasing Rio2 and phosphorylated Ltv1. We propose that conformational proofreading exerted via Rps20 constitutes a checkpoint permitting assembly factor release and progression of pre-40S maturation only after completion of all earlier maturation steps.

History

Deposition	Apr 10, 2019	-
Header (metadata) release	Jun 26, 2019	-
Map release	Jun 26, 2019	-
Update	Nov 25, 2020	-
Current status	Nov 25, 2020	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_4794.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: C1-headonly S20Delta loop yeast pre-40S particle Structural state 1 3D reconstruction of the head only(signal subtraction of the body) Local sharpening with LocScale
• Voxel size: X=Y=Z: 1.067 Å

Density

Contour Level	By AUTHOR: 0.09 / Movie #1: 0.09
Minimum - Maximum	-0.1706737 - 0.49626243
Average (Standard dev.)	0.001242879 (±0.012267725)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 384 384 384 Spacing 384 384 384
Cell	A=B=C: 409.72803 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.067	1.067	1.067
M x/y/z	384	384	384
origin x/y/z	0.000	0.000	0.000
length x/y/z	409.728	409.728	409.728
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	0
NX/NY/NZ	320	320	320
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	384	384	384
D min/max/mean	-0.171	0.496	0.001

Supplemental data

Half map: C1-headonly S20Delta loop yeast pre-40S particle Structural state...

• File: emd_4794_half_map_1.map
• Annotation: C1-headonly S20Delta loop yeast pre-40S particle Structural state 1 3D reconstruction of the head only(signal subtraction of the body) unfiltered half map 1

Half map: C1-headonly S20Delta loop yeast pre-40S particle Structural state...

• File: emd_4794_half_map_2.map
• Annotation: C1-headonly S20Delta loop yeast pre-40S particle Structural state 1 3D reconstruction of the head only(signal subtraction of the body) unfiltered half map 2

Sample components

Entire : C1-S20DeltaLoop State 1 of yeast pre-40S particles purified with ...

• Entire: Name: C1-S20DeltaLoop State 1 of yeast pre-40S particles purified with Tsr1-TAP as bait, in mutant conditions (deletion of aa 68-78 of Rps20) 3D reconstruction of the pre-40S head only (signal subtraction of the body domain)

Components

• Complex: C1-S20DeltaLoop State 1 of yeast pre-40S particles purified with Tsr1-TAP as bait, in mutant conditions (deletion of aa 68-78 of Rps20) 3D reconstruction of the pre-40S head only (signal subtraction of the body domain)

Supramolecule #1: C1-S20DeltaLoop State 1 of yeast pre-40S particles purified with ...

• Supramolecule: Name: C1-S20DeltaLoop State 1 of yeast pre-40S particles purified with Tsr1-TAP as bait, in mutant conditions (deletion of aa 68-78 of Rps20) 3D reconstruction of the pre-40S head only (signal subtraction of the body domain); type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#35
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast)
• Molecular weight: Theoretical: 1.2 MDa

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7.5
• Grid: Model: Quantifoil R2/1 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.045 kPa / Details: 25 mA
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: LEICA EM GP / Details: blot 1.7-1.9 seconds before plunging.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-25 / Number grids imaged: 1 / Number real images: 6480 / Average electron dose: 32.4 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 645109
• CTF correction: Software - Name: RELION (ver. 2.1)

Startup model

Type of model: PDB ENTRY
PDB model - PDB ID:

4v88

• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₁ (asymmetric) / Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 54130
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
• Final 3D classification: Software - Name: RELION

Atomic model buiding 1

Initial model

(PDB ID:

6fai

,

6eml

)

Output model

PDB-6rbd:
State 1 of yeast Tsr1-TAP Rps20-Deltaloop pre-40S particles

PDB-6rbe:
State 2 of yeast Tsr1-TAP Rps20-Deltaloop pre-40S particles