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- EMDB-44044: Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-Hfl... -

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Basic information

Entry
Database: EMDB / ID: EMD-44044
TitlePre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex
Map dataPre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex
Sample
  • Complex: Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex
Keywordsribosome splitting / Mycobacterium smegmatis 50S / HflX / TRANSLATION
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.96 Å
AuthorsMajumdar S / Koripella RK / Sharma MR / Manjari SR / Banavali NK / Agrawal RK
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01Al1554732 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI132422 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM061576 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: HflX-mediated drug resistance through ribosome splitting and rRNA disordering in mycobacteria.
Authors: Soneya Majumdar / Amuliya Kashyap / Ravi K Koripella / Manjuli R Sharma / Kelley Hurst-Hess / Swati R Manjari / Nilesh K Banavali / Pallavi Ghosh / Rajendra K Agrawal /
Abstract: HflX is a highly conserved ribosome-associated GTPase implicated in rescuing stalled ribosomes and mediating antibiotic resistance in several bacteria, including macrolide-lincosamide antibiotic ...HflX is a highly conserved ribosome-associated GTPase implicated in rescuing stalled ribosomes and mediating antibiotic resistance in several bacteria, including macrolide-lincosamide antibiotic resistance in mycobacteria. Mycobacterial HflXs carry a distinct N-terminal extension (NTE) and a small insertion, as compared to their eubacterial homologs. Here, we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the 70S ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX-mediated ribosome splitting and antibiotic resistance in mycobacteria. Our findings indicate that the NTE of mycobacterial HflX induces persistent disordering of multiple 23S rRNA helices, facilitating the dissociation of the 70S ribosome and generating an inactive pool of 50S subunits. During this process, HflX undergoes a large conformational change that stabilizes its NTE. Mycobacterial HflX also acts as an anti-association factor by binding to predissociated 50S subunits. Our structures show that a mycobacteria-specific insertion in HflX reaches far into the peptidyl transferase center (PTC), such that it would overlap with the ribosome-bound macrolide antibiotics. However, in the presence of antibiotics, this insertion retracts, adjusts around, and interacts with the antibiotic molecules. These results suggest that mycobacterial HflX is agnostic to antibiotic presence in the PTC. It mediates antibiotic resistance by splitting antibiotic-stalled 70S ribosomes and inactivating the resulting 50S subunits.
History
DepositionMar 12, 2024-
Header (metadata) releaseApr 30, 2025-
Map releaseApr 30, 2025-
UpdateApr 30, 2025-
Current statusApr 30, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44044.png

Downloads & links

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Map

FileDownload / File: emd_44044.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 400 pix.
= 500. Å		1.25 Å/pix.
x 400 pix.
= 500. Å		1.25 Å/pix.
x 400 pix.
= 500. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.25 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.144
Minimum - Maximum-0.18983594 - 0.5628151
Average (Standard dev.)0.0019252261 (±0.026553877)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 500.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex- half...

Fileemd_44044_half_map_1.map
AnnotationPre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex- half map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex- half...

Fileemd_44044_half_map_2.map
AnnotationPre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex- half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-Hfl...

EntireName: Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex
Components
  • Complex: Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex

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Supramolecule #1: Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-Hfl...

SupramoleculeName: Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
30.0 mMNH4ClAmmonium chloride
10.0 mMMgCl2Magnesium chloride
5.0 mMC2H6OSbeta-mercaptoethanol

Details: 20 mM HEPES-K at pH 7.5, 30 mM ammonium chloride, 10 mM magnesium chloride, and 5 mM beta-mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 3200FSC
TemperatureMin: 85.0 K / Max: 95.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Average electron dose: 21.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 120.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 5.2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 25000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 168372
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 20225
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: PROJECTION MATCHING
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2.15)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 48 / Target criteria: Cross-correlation coefficient

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