EMDB-12851: State D0'' of the human mitoribosomal large subunit assembly inte...

Basic information

• Entry: Database: EMDB / ID: EMD-12851
• Title: State D0'' of the human mitoribosomal large subunit assembly intermediate
• Map data: state D0''

Sample

• Complex: State D0'' of the human mitoribosomal large subunit assembly intermediate

Function / homology

Function:

tRNA (cytidine-5-)-methyltransferase activity / rRNA modification in the mitochondrion / negative regulation of mitochondrial translation / mitochondrial large ribosomal subunit assembly / rRNA (cytosine-C5-)-methyltransferase activity / Complex I biogenesis / protein lipoylation / negative regulation of ribosome biogenesis / Mitochondrial Fatty Acid Beta-Oxidation / Protein lipoylation / rRNA import into mitochondrion / mitochondrial [2Fe-2S] assembly complex / Respiratory electron transport / rRNA methyltransferase activity / mitochondrial transcription / mitochondrial translational termination / mitochondrial translational elongation / mitochondrial ribosome assembly / translation release factor activity, codon nonspecific / microprocessor complex / Mitochondrial translation elongation / Mitochondrial translation termination / Mitochondrial translation initiation / positive regulation of mitochondrial translation / protein targeting to mitochondrion / camera-type eye development / iron-sulfur cluster assembly complex / mitochondrial large ribosomal subunit / mitochondrial fission / mitochondrial large ribosomal subunit binding / peptidyl-tRNA hydrolase / mitochondrial ribosome / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / mitochondrial small ribosomal subunit / rRNA methylation / mitochondrial translation / aminoacyl-tRNA hydrolase activity / [2Fe-2S] cluster assembly / iron-sulfur cluster assembly / ribosomal large subunit binding / proton motive force-driven mitochondrial ATP synthesis / : / mitochondrial respiratory chain complex I assembly / mitochondrial electron transport, NADH to ubiquinone / respiratory chain complex I / acyl binding / anatomical structure morphogenesis / acyl carrier activity / RNA processing / Mitochondrial protein degradation / aerobic respiration / rescue of stalled ribosome / ribosomal large subunit biogenesis / Transferases; Transferring one-carbon groups; Methyltransferases / cellular response to leukemia inhibitory factor / methyltransferase activity / fatty acid binding / mitochondrial membrane / fibrillar center / fatty acid biosynthetic process / rRNA processing / double-stranded RNA binding / small ribosomal subunit rRNA binding / cell junction / heart development / large ribosomal subunit rRNA binding / 5S rRNA binding / double-stranded DNA binding / endonuclease activity / mitochondrial inner membrane / negative regulation of translation / nuclear body / rRNA binding / ribosome / structural constituent of ribosome / mitochondrial matrix / translation / ribonucleoprotein complex / protein domain specific binding / mRNA binding / nucleotide binding / calcium ion binding / synapse / regulation of DNA-templated transcription / nucleolus / apoptotic process / mitochondrion / RNA binding / extracellular space / nucleoplasm / nucleus / plasma membrane / cytosol

Domain/homology:

: / Mitochondrial termination factor repeats / Transcription termination factor, mitochondrial/chloroplastic / MTERF superfamily, mitochondrial/chloroplastic / mTERF / MIEF1-MP, LYR domain / Protein Iojap/ribosomal silencing factor RsfS / Ribosomal silencing factor during starvation / SAM-dependent methyltransferase RsmB/NOP2-type / RNA (C5-cytosine) methyltransferase / : / 16S rRNA methyltransferase RsmB/F / SAM-dependent MTase RsmB/NOP-type domain profile. / : / : / Ribosomal protein L55, mitochondrial / Mitochondrial ribosomal protein L48 / 39S ribosomal protein L40, mitochondrial / Mitochondrial ribosomal protein L55 superfamily / Mitochondrial ribosomal protein L55 / Ribosomal protein L37, mitochondrial / Mitochondrial ribosomal protein L46 NUDIX / Mitochondrial ribosomal protein L37 / Ribosomal protein S30, mitochondrial / Ribosomal protein L53, mitochondrial / 39S ribosomal protein L53/MRP-L53 / 39S ribosomal protein L42, mitochondrial / Mitochondrial 28S ribosomal protein S32 / Ribosomal protein L28/L40, mitochondrial / Mitochondrial ribosomal protein L28 / Ribosomal protein 63, mitochondrial / Growth arrest/ DNA-damage-inducible protein-interacting protein 1 / Ribosomal protein L51, mitochondrial / Growth arrest and DNA-damage-inducible proteins-interacting protein 1 / Mitochondrial ribosomal subunit / Mitochondrial ribosome protein 63 / Ribosomal protein L37/S30 / Growth arrest and DNA damage-inducible proteins-interacting protein 1 domain superfamily / : / : / Mitochondrial 28S ribosomal protein S30 (PDCD9) / 39S ribosomal protein L52, mitochondrial / Mitoribosomal protein mL52 / : / Ribosomal protein L35, mitochondrial / MRPL44, double-stranded RNA binding domain / MRPL44 dsRNA-binding domain / Large ribosomal subunit protein mL44, endonuclease domain / : / Large ribosomal subunit protein bL9m C-terminal domain / Tim44-like domain / Tim44-like domain / Tim44 / : / : / Ribosomal protein L49/IMG2 / Mitochondrial large subunit ribosomal protein (Img2) / Ribosomal protein L46, N-terminal / 39S mitochondrial ribosomal protein L46 / Ribosomal protein L50, mitochondria / Ribosomal protein L27/L41, mitochondrial / Mitochondrial ribosomal protein L27 / Ribosomal subunit 39S / 39S ribosomal protein L46, mitochondrial / : / 39S ribosomal protein L43/54S ribosomal protein L51 / Ribosomal protein L47, mitochondrial / MRP-L47 superfamily, mitochondrial / Mitochondrial 39-S ribosomal protein L47 (MRP-L47) / Phosphatidylethanolamine-binding protein / Phosphatidylethanolamine-binding protein / Phosphatidylethanolamine-binding protein, eukaryotic / PEBP-like superfamily / Threonyl/alanyl tRNA synthetase, class II-like, putative editing domain superfamily / TGS-like / TGS domain profile. / TGS / Peptide chain release factor class I / RF-1 domain / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / Complex 1 LYR protein domain / Complex 1 protein (LYR family) / : / Beta-grasp domain superfamily / Ribosomal protein/NADH dehydrogenase domain / Mitochondrial ribosomal protein L51 / S25 / CI-B8 domain / Mitochondrial ribosomal protein L51 / S25 / CI-B8 domain / Acyl carrier protein (ACP) / NTF2-like domain superfamily / NUDIX hydrolase-like domain superfamily / Ribosomal protein L11, bacterial-type / Phosphopantetheine attachment site / Nucleotidyltransferase superfamily / Phosphopantetheine attachment site. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / Phosphopantetheine attachment site

Component:

Mitochondrial ribosome and complex I assembly factor AltMIEF1 / Acyl carrier protein, mitochondrial / Large ribosomal subunit protein bL33m / Large ribosomal subunit protein uL3m / Large ribosomal subunit protein bL19m / Large ribosomal subunit protein bL28m / Large ribosomal subunit protein mL49 / Large ribosomal subunit protein mL62 / Large ribosomal subunit protein uL23m / Large ribosomal subunit protein mL51 / Large ribosomal subunit protein uL2m / Large ribosomal subunit protein mL54 / Large ribosomal subunit protein uL14m / Large ribosomal subunit protein bL21m / Transcription termination factor 4, mitochondrial / Large ribosomal subunit protein mL55 / Large ribosomal subunit protein uL10m / Large ribosomal subunit protein mL52 / Large ribosomal subunit protein mL41 / Large ribosomal subunit protein mL50 / Large ribosomal subunit protein mL43 / Large ribosomal subunit protein mL64 / Large ribosomal subunit protein uL30m / Large ribosomal subunit protein uL24m / 5-methylcytosine rRNA methyltransferase NSUN4 / Large ribosomal subunit protein mL38 / Mitochondrial assembly of ribosomal large subunit protein 1 / Large ribosomal subunit protein mL53 / Large ribosomal subunit protein mL48 / Large ribosomal subunit protein bL34m / Large ribosomal subunit protein mL63 / Large ribosomal subunit protein mL45 / Large ribosomal subunit protein bL32m / Large ribosomal subunit protein bL20m / Large ribosomal subunit protein uL13m / Large ribosomal subunit protein bL9m / Large ribosomal subunit protein uL4m / Large ribosomal subunit protein mL37 / Large ribosomal subunit protein uL18m / Large ribosomal subunit protein mL46 / Large ribosomal subunit protein mL44 / Large ribosomal subunit protein uL29m / Large ribosomal subunit protein mL65 / Large ribosomal subunit protein mL40 / Large ribosomal subunit protein bL17m / Large ribosomal subunit protein mL66 / Large ribosomal subunit protein uL22m / Large ribosomal subunit protein uL16m / Large ribosomal subunit protein mL39 / Large ribosomal subunit protein bL35m / Large ribosomal subunit protein uL15m / Large ribosomal subunit protein bL36m / Large ribosomal subunit protein bL27m / Large ribosomal subunit protein uL11m / Large ribosomal subunit protein mL42

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.5 Å
• Authors: Lenarcic T / Jaskolowski M / Leibundgut M / Scaiola A / Schoenhut T / Saurer M / Lee RG / Rackham O / Filipovska A / Ban N

Funding support

Switzerland, 3 items

Organization	Grant number	Country
Swiss National Science Foundation	31003A_182341	Switzerland
European Molecular Biology Organization (EMBO)	1074-2019
Swiss National Science Foundation	182880

• Citation: Journal: Nat Commun / Year: 2021; Title: Stepwise maturation of the peptidyl transferase region of human mitoribosomes.; Authors: Tea Lenarčič / Mateusz Jaskolowski / Marc Leibundgut / Alain Scaiola / Tanja Schönhut / Martin Saurer / Richard G Lee / Oliver Rackham / Aleksandra Filipovska / Nenad Ban / ; Abstract: Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes have diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. The structural basis of the mammalian mitochondrial ribosome assembly is currently not well understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving seven assembly factors. We discover that the NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by the MRM2 methyltransferase and quality control interactions with the conserved mitochondrial GTPase MTG2 that contacts the sarcin-ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.

History

Deposition	Apr 30, 2021	-
Header (metadata) release	Jun 23, 2021	-
Map release	Jun 23, 2021	-
Update	Jul 7, 2021	-
Current status	Jul 7, 2021	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_12851.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: state D0''
• Voxel size: X=Y=Z: 1.06 Å

Density

Contour Level	By AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum	-0.8602798 - 2.1715112
Average (Standard dev.)	0.005818044 (±0.091339305)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 448 448 448 Spacing 448 448 448
Cell	A=B=C: 474.87997 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.06	1.06	1.06
M x/y/z	448	448	448
origin x/y/z	0.000	0.000	0.000
length x/y/z	474.880	474.880	474.880
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	192	139	186
NX/NY/NZ	211	274	246
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	448	448	448
D min/max/mean	-0.860	2.172	0.006

Supplemental data

Sample components

Entire : State D0'' of the human mitoribosomal large subunit assembly inte...

• Entire: Name: State D0'' of the human mitoribosomal large subunit assembly intermediate

Components

• Complex: State D0'' of the human mitoribosomal large subunit assembly intermediate

Supramolecule #1: State D0'' of the human mitoribosomal large subunit assembly inte...

• Supramolecule: Name: State D0'' of the human mitoribosomal large subunit assembly intermediate; type: complex / ID: 1 / Parent: 0
• Source (natural): Organism: Homo sapiens (human) / Strain: HEK 293 EBNA

Experimental details

Structure determination

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

Sample preparation

Buffer

pH: 7.6
Component:

Concentration	Formula
20.0 mM	HEPES-KOH
100.0 mM	KCl
20.0 mM	MgCl2
1.0 mM	DTT

• Grid: Model: Quantifoil R2/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS
• Vitrification: Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV; Details: 4 uL of the sample was blotted for 2-6 sec before plunging.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• 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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• CTF correction: Software - Name: cryoSPARC (ver. 3.1)
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 32871
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)