EMDB-10176: mt-SSU assemblosome of wild-type Trypanosoma brucei brucei

Basic information

• Entry: Database: EMDB / ID: EMD-10176
• Title: mt-SSU assemblosome of wild-type Trypanosoma brucei brucei
• Map data: mt-SSU assemblosome of wild-type Trypanosoma brucei brucei

Sample

• Complex: wild-type mt-SSU assemblosome

Function / homology

Function:

modulation of formation of structure involved in a symbiotic process / rRNA (cytosine-N4-)-methyltransferase activity / mitochondrial mRNA editing complex / mitochondrial RNA processing / ATP-activated inward rectifier potassium channel activity / kinetoplast / thiosulfate-cyanide sulfurtransferase activity / quorum sensing / response to arsenic-containing substance / rRNA base methylation / ciliary plasm / nuclear lumen / mRNA stabilization / mitochondrial small ribosomal subunit / acyl binding / acyl carrier activity / iron-sulfur cluster binding / voltage-gated potassium channel activity / voltage-gated potassium channel complex / Transferases; Transferring one-carbon groups; Methyltransferases / methyltransferase activity / potassium ion transport / protein homooligomerization / fatty acid biosynthetic process / structural constituent of ribosome / mitochondrial matrix / translation / mRNA binding / mitochondrion / RNA binding / membrane / metal ion binding / cytosol / cytoplasm

Domain/homology:

T-cell activation inhibitor, mitochondrial / Domain of unknown function DUF4460 / Domain of unknown function (DUF4460) / Mitochondrial glycoprotein / Mitochondrial glycoprotein superfamily / Mitochondrial glycoprotein / Probable Zinc-ribbon domain / Probable Treble clef zinc finger domain / Ribosomal RNA small subunit methyltransferase H / S-adenosyl-L-methionine-dependent methyltransferase, MraW, recognition domain superfamily / MraW methylase family / Ribosomal protein Rsm22-like / : / Mitochondrial small ribosomal subunit Rsm22 / Protein Fyv4 / IGR protein motif / IGR / Phosphotyrosine protein phosphatase I / Phosphotyrosine protein phosphatase I superfamily / Low molecular weight phosphotyrosine protein phosphatase / Low molecular weight phosphatase family / Pentatricopeptide repeat domain / Pentatricopeptide (PPR) repeat profile. / Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Ribosomal protein S23/S29, mitochondrial / Mitochondrial ribosomal death-associated protein 3 / Pentatricopeptide repeat / Rhodanese-like domain / Rhodanese domain profile. / Rhodanese-like domain superfamily / Rhodanese-like domain / Acyl carrier protein (ACP) / Phosphopantetheine attachment site / SKP1/BTB/POZ domain superfamily / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Translation elongation factor EF1B/ribosomal protein S6 / Tetratricopeptide-like helical domain superfamily / Ribosomal protein S9 / Ribosomal protein S9/S16 / Ribosomal protein S11 superfamily / S15/NS1, RNA-binding / Thioredoxin-like superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / S-adenosyl-L-methionine-dependent methyltransferase superfamily

Component:

Uncharacterized protein / Uncharacterized protein / Uncharacterized protein / Methyltransferase / Mitochondrial SSU ribosomal protein / Uncharacterized protein / Mitochondrial SSU ribosomal protein / Uncharacterized protein / Potassium voltage-gated channel, putative / Uncharacterized protein / Small ribosomal subunit protein mS41 / Uncharacterized protein / Uncharacterized protein / Uncharacterized protein / Uncharacterized protein / Uncharacterized protein / Target SNARE / Phosphotyrosine protein phosphatase I domain-containing protein / DUF4460 domain-containing protein / Uncharacterized protein / Uncharacterized protein / Pentacotripeptide-repeat region of PRORP domain-containing protein / Uncharacterized protein / Uncharacterized protein / Glutaredoxin domain-containing protein / S-adenosyl-methyltransferase mraW-like protein, putative / Ribosomal protein S9 / Uncharacterized protein / Acyl carrier protein / Mitochondrial glycoprotein-like protein / Uncharacterized protein / Pentacotripeptide-repeat region of PRORP domain-containing protein / Uncharacterized protein / Probable Zinc-ribbon domain-containing protein / Succinate dehydrogenase assembly factor 3, mitochondrial / Uncharacterized protein / Uncharacterized protein / Small ribosomal subunit protein mS29 / Rhodanese domain-containing protein / Uncharacterized protein

• Biological species: Trypanosoma brucei brucei (eukaryote)
• Method: single particle reconstruction / cryo EM / Resolution: 4.0 Å
• Authors: Saurer M / Ramrath D / Niemann M / Calderaro S / Prange C / Mattei S / Scaiola A / Leitner A / Bieri P / Horn EK / Leibundgut M / Schneider A / Boehringer D / Ban N

Funding support

Switzerland, 1 items

Organization	Grant number	Country
Swiss National Science Foundation		Switzerland

• Citation: Journal: Science / Year: 2019; Title: Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery.; Authors: Martin Saurer / David J F Ramrath / Moritz Niemann / Salvatore Calderaro / Céline Prange / Simone Mattei / Alain Scaiola / Alexander Leitner / Philipp Bieri / Elke K Horn / Marc Leibundgut / Daniel Boehringer / André Schneider / Nenad Ban / ; Abstract: Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo-electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.

History

Deposition	Aug 3, 2019	-
Header (metadata) release	Sep 25, 2019	-
Map release	Sep 25, 2019	-
Update	Sep 25, 2019	-
Current status	Sep 25, 2019	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_10176.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: mt-SSU assemblosome of wild-type Trypanosoma brucei brucei
• Voxel size: X=Y=Z: 1.5318 Å

Density

Contour Level	By AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum	-0.14604793 - 0.32192507
Average (Standard dev.)	0.0009849102 (±0.008526604)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 340 340 340 Spacing 340 340 340
Cell	A=B=C: 520.812 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.5318	1.5318	1.5318
M x/y/z	340	340	340
origin x/y/z	0.000	0.000	0.000
length x/y/z	520.812	520.812	520.812
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	340	340	340
D min/max/mean	-0.146	0.322	0.001

Supplemental data

Sample components

Entire : wild-type mt-SSU assemblosome

• Entire: Name: wild-type mt-SSU assemblosome

Components

• Complex: wild-type mt-SSU assemblosome

Supramolecule #1: wild-type mt-SSU assemblosome

• Supramolecule: Name: wild-type mt-SSU assemblosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#103
• Source (natural): Organism: Trypanosoma brucei brucei (eukaryote) / Strain: Lister strain 427

Experimental details

Structure determination

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

Sample preparation

• Buffer: pH: 7.4
• Grid: Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 15 mA
• Vitrification: Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
• Details: Mitochondrial ribosomal small subunit assemblosome of wild-type Trypanosoma brucei brucei Lister strain 427

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 13503 / Average exposure time: 1.5 sec. / Average electron dose: 75.0 e/Ų; Details: Images were collected in movie-mode at 40 frames per second
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.5 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 129032 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 75000
• 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: 1243432
• CTF correction: Software - Name: Gctf (ver. 1.06) / Details: On the fly in RELION
• Startup model: Type of model: NONE; Details: The starting model was determined ab-initio using RELION 3D initial model routine based on SGD.
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.5) / Number images used: 28663
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)

Atomic model buiding 1

• Details: Starting models of ribosomal proteins of the T. brucei mitochondrial ribosomal small subunit were taken from PDB 6HIW. Mitchondrial ribosomal small subunit assembly factors were built de novo.
• Refinement: Protocol: OTHER