Journal: Nucleic Acids Res / Year: 2016 Title: YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit. Authors: Xiaodan Ni / Joseph H Davis / Nikhil Jain / Aida Razi / Samir Benlekbir / Andrew G McArthur / John L Rubinstein / Robert A Britton / James R Williamson / Joaquin Ortega / Abstract: YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we ...YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5-6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit.
History
Deposition
Jun 30, 2016
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Header (metadata) release
Jul 13, 2016
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Map release
Aug 17, 2016
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Update
Oct 12, 2016
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Current status
Oct 12, 2016
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_8275.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Assembly intermediate 50S subunit
Voxel size
X=Y=Z: 1.45 Å
Density
Contour Level
By AUTHOR: 0.0185 / Movie #1: 0.0185
Minimum - Maximum
-0.010335746 - 0.0570948
Average (Standard dev.)
0.0009669719 (±0.004562654)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
240
240
240
Spacing
240
240
240
Cell
A=B=C: 348.0 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.45
1.45
1.45
M x/y/z
240
240
240
origin x/y/z
0.000
0.000
0.000
length x/y/z
348.000
348.000
348.000
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-15
2
-37
NX/NY/NZ
99
82
71
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
240
240
240
D min/max/mean
-0.010
0.057
0.001
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Supplemental data
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Sample components
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Entire : Assembly intermediate of the 50S ribosomal subunit, Class II
Entire
Name: Assembly intermediate of the 50S ribosomal subunit, Class II
Components
Complex: Assembly intermediate of the 50S ribosomal subunit, Class II
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Supramolecule #1: Assembly intermediate of the 50S ribosomal subunit, Class II
Supramolecule
Name: Assembly intermediate of the 50S ribosomal subunit, Class II type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Obtained upon depletion of YsxC protein
Source (natural)
Organism: Bacillus subtilis (bacteria)
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.5
Grid
Model: C-flats CFT-222C / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK III Details: Plunged into liquid ethane (FEI VITROBOT MARK III).
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Electron microscopy
Microscope
FEI TECNAI F20
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 0.5 sec. / Average electron dose: 1.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
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Image processing
Particle selection
Number selected: 87684
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 46430
Initial angle assignment
Type: PROJECTION MATCHING
Final angle assignment
Type: PROJECTION MATCHING
FSC plot (resolution estimation)
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