EMDB-12760: Disome of rabbit ribosomes with the leading one stalled by the SA...

Basic information

• Entry: Database: EMDB / ID: EMD-12760
• Title: Disome of rabbit ribosomes with the leading one stalled by the SARS-CoV-2 pseudoknot
• Map data: Composite map

Sample

• Complex: Disome of rabbit ribosomes with the leading one stalled by the SARS-CoV-2 pseudoknot

Function / homology

Function:

Major pathway of rRNA processing in the nucleolus and cytosol / GTP hydrolysis and joining of the 60S ribosomal subunit / L13a-mediated translational silencing of Ceruloplasmin expression / SRP-dependent cotranslational protein targeting to membrane / Formation of a pool of free 40S subunits / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / ribosomal subunit / laminin receptor activity / regulation of G1 to G0 transition / exit from mitosis / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / regulation of translation involved in cellular response to UV / protein-DNA complex disassembly / positive regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / optic nerve development / mammalian oogenesis stage / retinal ganglion cell axon guidance / G1 to G0 transition / activation-induced cell death of T cells / positive regulation of signal transduction by p53 class mediator / ubiquitin ligase inhibitor activity / phagocytic cup / TOR signaling / T cell proliferation involved in immune response / protein-RNA complex assembly / ribosomal small subunit export from nucleus / erythrocyte development / translation regulator activity / cellular response to actinomycin D / cytosolic ribosome / laminin binding / rough endoplasmic reticulum / gastrulation / MDM2/MDM4 family protein binding / DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest / class I DNA-(apurinic or apyrimidinic site) endonuclease activity / DNA-(apurinic or apyrimidinic site) lyase / negative regulation of ubiquitin-dependent protein catabolic process / rescue of stalled ribosome / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / 90S preribosome / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA / ribosomal large subunit biogenesis / cellular response to leukemia inhibitory factor / maturation of SSU-rRNA / positive regulation of translation / small-subunit processome / positive regulation of apoptotic signaling pathway / protein kinase C binding / positive regulation of protein-containing complex assembly / placenta development / cellular response to gamma radiation / mRNA 5'-UTR binding / transcription coactivator binding / cytoplasmic ribonucleoprotein granule / modification-dependent protein catabolic process / spindle / G1/S transition of mitotic cell cycle / rRNA processing / antimicrobial humoral immune response mediated by antimicrobial peptide / protein tag activity / rhythmic process / positive regulation of canonical Wnt signaling pathway / protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / ribosome biogenesis / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / ribosome binding / glucose homeostasis / retina development in camera-type eye / Lyases; Phosphorus-oxygen lyases / virus receptor activity / regulation of translation / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ribosomal small subunit biogenesis / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / ribosomal small subunit assembly / TRAF3-dependent IRF activation pathway / Replication of the SARS-CoV-2 genome / snRNP Assembly / double membrane vesicle viral factory outer membrane / small ribosomal subunit / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / small ribosomal subunit rRNA binding / 5'-3' DNA helicase activity / SARS coronavirus main proteinase / host cell endosome / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / cell body / symbiont-mediated suppression of host toll-like receptor signaling pathway / T cell differentiation in thymus / symbiont-mediated degradation of host mRNA

Domain/homology:

60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / 40S ribosomal protein SA / 40S ribosomal protein SA, C-terminal domain / 40S ribosomal protein SA C-terminus / Ubiquitin-like protein FUBI / Ribosomal protein L6, N-terminal / Ribosomal protein L6, N-terminal domain / Ribosomal protein L30e / Ribosomal L15/L27a, N-terminal / Ribosomal protein L23 / 50S ribosomal protein L10, insertion domain superfamily / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / : / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S12e signature. / metallochaperone-like domain / Ribosomal protein S12e / TRASH domain / Small (40S) ribosomal subunit Asc1/RACK1 / Ribosomal protein S2, eukaryotic / Ribosomal protein L29e / Ribosomal L29e protein family / 40S Ribosomal protein S10 / S27a-like superfamily / Ribosomal protein S10, eukaryotic/archaeal / Ribosomal protein L1, conserved site / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal protein L1 signature. / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal L38e protein family / Ribosomal protein S2, eukaryotic/archaeal / : / Ribosomal protein L1 / Ribosomal protein S17e, conserved site / Ribosomal protein S17e signature. / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein S30 / Ribosomal protein S30 / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein S7e signature. / Ribosomal protein L44e signature. / Ribosomal protein L10e, conserved site / Ribosomal protein L10e signature. / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein L10e / Ribosomal protein L19, eukaryotic / Ribosomal protein S3Ae, conserved site / Ribosomal protein S3Ae signature. / Ribosomal protein S27e signature. / : / Ribosomal protein S19A/S15e / Ribosomal protein S8e, conserved site / Ribosomal protein S8e signature. / Ribosomal protein L24e, conserved site / Ribosomal protein L24e signature. / Ribosomal protein L44e / Ribosomal protein L19/L19e conserved site / Ribosomal protein L44 / Ribosomal protein L19e signature. / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein S17e / Ribosomal protein S17e-like superfamily / Ribosomal S17 / Ribosomal protein L6e signature. / Ribosomal protein L30e signature 1. / Ribosomal protein S6, eukaryotic / 40S ribosomal protein S1/3, eukaryotes / Ribosomal S24e conserved site / Ribosomal protein S24e signature. / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein L23/L25, N-terminal / Ribosomal protein L23, N-terminal domain / Ribosomal protein L30e signature 2. / 40S ribosomal protein S11, N-terminal / Ribosomal_S17 N-terminal / Ribosomal protein S7e / Ribosomal protein L30e, conserved site / Ribosomal protein S7e / Ribosomal protein L27e / Ribosomal protein L27e superfamily / Ribosomal L27e protein family / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L36e signature. / Ribosomal protein L39e, conserved site / Ribosomal protein L39e signature.

Component:

Small ribosomal subunit protein eS32 / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein uS4 / Large ribosomal subunit protein eL24 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL33 / Small ribosomal subunit protein eS12 / Large ribosomal subunit protein eL29 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein eL31 / Large ribosomal subunit protein eL21 / Large ribosomal subunit protein uL29 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein RACK1 / Ubiquitin-ribosomal protein eS31 fusion protein / Large ribosomal subunit protein eL6 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL24 / Small ribosomal subunit protein eS1 / Small ribosomal subunit protein eS7 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein eL43 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein eL14 / Small ribosomal subunit protein uS12 / Large ribosomal subunit protein eL15 / Small ribosomal subunit protein uS11 / 40S ribosomal protein S24 / Large ribosomal subunit protein uL14 / Ubiquitin-like FUBI-ribosomal protein eS30 fusion protein / Small ribosomal subunit protein eS25 / Large ribosomal subunit protein eL30 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein eS28 / Small ribosomal subunit protein eS8 / Large ribosomal subunit protein uL3 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein eS6 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein eS10 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein eL36 / Small ribosomal subunit protein eS17 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein eL27 / Large ribosomal subunit protein eL34 / Small ribosomal subunit protein eS27 / Large ribosomal subunit protein eL38 / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein eL42 / Large ribosomal subunit protein eL32 / Small ribosomal subunit protein uS14 / Replicase polyprotein 1ab / Large ribosomal subunit protein eL19 / Large ribosomal subunit protein eL37

• Biological species: Oryctolagus cuniculus (rabbit)
• Method: single particle reconstruction / cryo EM / Resolution: 6.0 Å
• Authors: Bhatt PR / Scaiola A / Leibundgut MA / Atkins JF / Ban N

Funding support

Switzerland, Ireland, 2 items

Organization	Grant number	Country
Swiss National Science Foundation	31003A_182341	Switzerland
Irish Research Council	IRCLA/2019/74	Ireland

• Citation: Journal: Science / Year: 2021; Title: Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome.; Authors: Pramod R Bhatt / Alain Scaiola / Gary Loughran / Marc Leibundgut / Annika Kratzel / Romane Meurs / René Dreos / Kate M O'Connor / Angus McMillan / Jeffrey W Bode / Volker Thiel / David Gatfield / John F Atkins / Nenad Ban / ; Abstract: Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

History

Deposition	Apr 14, 2021	-
Header (metadata) release	Jun 2, 2021	-
Map release	Jun 2, 2021	-
Update	Jul 7, 2021	-
Current status	Jul 7, 2021	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_12760.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Composite map
• Voxel size: X=Y=Z: 2.98953 Å

Density

Contour Level	By AUTHOR: 0.7 / Movie #1: 1.1
Minimum - Maximum	-1.7641442 - 5.231067
Average (Standard dev.)	0.030923454 (±0.24414854)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 765.3197 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	2.98953125	2.98953125	2.98953125
M x/y/z	256	256	256
origin x/y/z	0.000	0.000	0.000
length x/y/z	765.320	765.320	765.320
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	0
NX/NY/NZ	280	280	280
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	256	256	256
D min/max/mean	-1.764	5.231	0.031

Supplemental data

Mask #1

• File: emd_12760_msk_1.map

Mask #2

• File: emd_12760_msk_2.map

Additional map: #2

• File: emd_12760_additional_1.map

Additional map: #1

• File: emd_12760_additional_2.map

Sample components

Entire : Disome of rabbit ribosomes with the leading one stalled by the SA...

• Entire: Name: Disome of rabbit ribosomes with the leading one stalled by the SARS-CoV-2 pseudoknot

Components

• Complex: Disome of rabbit ribosomes with the leading one stalled by the SARS-CoV-2 pseudoknot

Supramolecule #1: Disome of rabbit ribosomes with the leading one stalled by the SA...

• Supramolecule: Name: Disome of rabbit ribosomes with the leading one stalled by the SARS-CoV-2 pseudoknot; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#86
• Source (natural): Organism: Oryctolagus cuniculus (rabbit)

Experimental details

Structure determination

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

Sample preparation

• Concentration: 0.3 mg/mL
• Buffer: pH: 7.4
• Grid: Model: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 1.0 nm
• Vitrification: Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 100.0 µm / Calibrated magnification: 56604 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 81000
• 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

• CTF correction: Software - Name: Gctf
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 19009
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)