EMDB-21480: Class 3 from 3D classification of Bacillus subtilus delta HPF rib...

Basic information

• Entry: Database: EMDB / ID: EMD-21480
• Title: Class 3 from 3D classification of Bacillus subtilus delta HPF ribosomes
• Map data: Class 3 from 3D classification of Bacillus subtilus delta HPF ribosomes

Sample

• Complex: Delta hpf 70S

• Biological species: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
• Method: single particle reconstruction / cryo EM / Resolution: 4.9 Å
• Authors: Feaga HA / Kopylov M / Jenny KK / Marko J / Dworkin J

Funding support

United States, 1 items

Organization	Grant number	Country
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	R01 GM114213	United States

• Citation: Journal: J Bacteriol / Year: 2020; Title: Ribosome Dimerization Protects the Small Subunit.; Authors: Heather A Feaga / Mykhailo Kopylov / Jenny Kim Kim / Marko Jovanovic / Jonathan Dworkin / ; Abstract: When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to favorable conditions. Here, we show that the ribosome dimerization protein hibernation-promoting factor (HPF) functions to protect essential ribosomal proteins. Ribosomes isolated from strains lacking HPF (Δ) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located directly at the ribosome dimer interface. We used single-particle cryo-electron microscopy (cryo-EM) to further characterize these ribosomes and observed that a high percentage of ribosomes were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function. HPF is almost universally conserved in bacteria, and HPF deletions in diverse species exhibit decreased viability during starvation. Our data provide mechanistic insight into this phenotype and establish a mechanism for how HPF protects ribosomes during quiescence. The formation of ribosome dimers during periods of dormancy is widespread among bacteria. Dimerization is typically mediated by a single protein, hibernation-promoting factor (HPF). Bacteria lacking HPF exhibit strong defects in viability and pathogenesis and, in some species, extreme loss of rRNA. The mechanistic basis of these phenotypes has not been determined. Here, we report that HPF from the Gram-positive bacterium preserves ribosomes by preventing the loss of essential ribosomal proteins at the dimer interface. This protection may explain phenotypes associated with the loss of HPF, since ribosome protection would aid survival during nutrient limitation and impart a strong selective advantage when the bacterial cell rapidly reinitiates growth in the presence of sufficient nutrients.

History

Deposition	Feb 27, 2020	-
Header (metadata) release	Mar 25, 2020	-
Map release	Mar 25, 2020	-
Update	May 13, 2020	-
Current status	May 13, 2020	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_21480.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Class 3 from 3D classification of Bacillus subtilus delta HPF ribosomes
• Voxel size: X=Y=Z: 2.121 Å

Density

Contour Level	By AUTHOR: 1 / Movie #1: 1
Minimum - Maximum	-0.6995345 - 3.1978517
Average (Standard dev.)	0.06810672 (±0.28311267)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 192 192 192 Spacing 192 192 192
Cell	A=B=C: 407.232 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	2.121	2.121	2.121
M x/y/z	192	192	192
origin x/y/z	0.000	0.000	0.000
length x/y/z	407.232	407.232	407.232
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	192	192	192
D min/max/mean	-0.700	3.198	0.068

Supplemental data

Sample components

Entire : Delta hpf 70S

• Entire: Name: Delta hpf 70S

Components

• Complex: Delta hpf 70S

Supramolecule #1: Delta hpf 70S

• Supramolecule: Name: Delta hpf 70S / type: complex / ID: 1 / Parent: 0
• Source (natural): Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)

Experimental details

Structure determination

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

Sample preparation

• Buffer: pH: 7.4
• Grid: Model: UltrAuFoil / Support film - Material: CARBON / Support film - topology: HOLEY
• Vitrification: Cryogen name: ETHANE

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
• Sample stage: Cooling holder cryogen: NITROGEN
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 56.7 e/Ų
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Initial angle assignment: Type: RANDOM ASSIGNMENT
• Final angle assignment: Type: MAXIMUM LIKELIHOOD
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 13692