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- EMDB-48574: Mycobacterial open-gate proteasome in complex with engineered NtrC1 -

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Basic information

Entry
Database: EMDB / ID: EMD-48574
TitleMycobacterial open-gate proteasome in complex with engineered NtrC1
Map dataunsharpened map
Sample
  • Complex: Mycobacterial open-gate proteasome capped with engineered bacterial enhancer-binding protein NtrC1 in presence of ADP-AlFx
    • Protein or peptide: NtrC1-GS4
    • Protein or peptide: mycobacterial proteasome subunit alpha
    • Protein or peptide: mycobacterial proteasome subunit beta
Keywordssigma N / sigma 54 / ATPase / bacterial enhancer binding protein / transcription initiation / intermediate / TRANSCRIPTION
Function / homology
Function and homology information


symbiont-mediated perturbation of host defenses / zymogen binding / DNA-binding transcription activator activity / phosphorelay signal transduction system / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / cis-regulatory region sequence-specific DNA binding ...symbiont-mediated perturbation of host defenses / zymogen binding / DNA-binding transcription activator activity / phosphorelay signal transduction system / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / cis-regulatory region sequence-specific DNA binding / proteolysis involved in protein catabolic process / peptidoglycan-based cell wall / protein-DNA complex / modification-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / positive regulation of DNA-templated transcription / extracellular region / ATP binding / metal ion binding / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
AAA+ ATPase lid domain / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / Sigma-54 interaction domain ATP-binding region A signature. / Sigma-54 interaction domain, ATP-binding site 1 / Sigma-54 interaction domain, ATP-binding site 2 / Sigma-54 interaction domain ATP-binding region B signature. / Sigma-54 interaction domain profile. / Sigma-54 interaction domain / RNA polymerase sigma factor 54 interaction domain ...AAA+ ATPase lid domain / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / Sigma-54 interaction domain ATP-binding region A signature. / Sigma-54 interaction domain, ATP-binding site 1 / Sigma-54 interaction domain, ATP-binding site 2 / Sigma-54 interaction domain ATP-binding region B signature. / Sigma-54 interaction domain profile. / Sigma-54 interaction domain / RNA polymerase sigma factor 54 interaction domain / DNA binding HTH domain, Fis-type / Bacterial regulatory protein, Fis family / Response regulator receiver domain / cheY-homologous receiver domain / Signal transduction response regulator, receiver domain / Response regulatory domain profile. / CheY-like superfamily / Proteasome B-type subunit / Proteasome beta-type subunit profile. / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal / Homeobox-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Transcriptional regulator (NtrC family) / Proteasome subunit beta / Proteasome subunit alpha
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Aquifex aeolicus VF5 (bacteria) / Mycobacterium tuberculosis H37Rv (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsMolina N / Mueller AU / Darst SA
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118130 United States
Life Sciences Research FoundationAgouron Institute United States
CitationJournal: Nat Commun / Year: 2025
Title: Real-time capture of σ transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding.
Authors: Andreas U Mueller / Nina Molina / B Tracy Nixon / Seth A Darst /
Abstract: Bacterial σ factors bind RNA polymerase (E) to form holoenzyme (Eσ), conferring promoter specificity to E and playing a key role in transcription bubble formation. σ is unique among σ factors in ...Bacterial σ factors bind RNA polymerase (E) to form holoenzyme (Eσ), conferring promoter specificity to E and playing a key role in transcription bubble formation. σ is unique among σ factors in its structure and functional mechanism, requiring activation by specialized AAA+ ATPases. Eσ forms an inactive promoter complex where the N-terminal σ region I (σ-RI) threads through a small DNA bubble. On the opposite side of the DNA, the ATPase engages σ-RI within the pore of its hexameric ring. Here, we perform kinetics-guided structural analysis of de novo formed Eσ initiation complexes and engineer a biochemical assay to measure ATPase-mediated σ-RI translocation during promoter melting. We show that the ATPase exerts mechanical action to translocate about 30 residues of σ-RI through the DNA bubble, disrupting inhibitory structures of σ to allow full transcription bubble formation. A local charge switch of σ-RI from positive to negative may help facilitate disengagement of the otherwise processive ATPase, allowing subsequent σ disentanglement from the DNA bubble.
History
DepositionJan 9, 2025-
Header (metadata) releaseAug 13, 2025-
Map releaseAug 13, 2025-
UpdateAug 13, 2025-
Current statusAug 13, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48574.png

Downloads & links

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Map

FileDownload / File: emd_48574.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.5 Å/pix.
x 288 pix.
= 432. Å		1.5 Å/pix.
x 288 pix.
= 432. Å		1.5 Å/pix.
x 288 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.45757246 - 1.2048037
Average (Standard dev.)-0.0051022535 (±0.0657228)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 432.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48574_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Additional map: sharpened map (b-factor 103.3)

Fileemd_48574_additional_1.map
Annotationsharpened map (b-factor 103.3)
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_48574_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_48574_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Mycobacterial open-gate proteasome capped with engineered bacteri...

EntireName: Mycobacterial open-gate proteasome capped with engineered bacterial enhancer-binding protein NtrC1 in presence of ADP-AlFx
Components
  • Complex: Mycobacterial open-gate proteasome capped with engineered bacterial enhancer-binding protein NtrC1 in presence of ADP-AlFx
    • Protein or peptide: NtrC1-GS4
    • Protein or peptide: mycobacterial proteasome subunit alpha
    • Protein or peptide: mycobacterial proteasome subunit beta

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Supramolecule #1: Mycobacterial open-gate proteasome capped with engineered bacteri...

SupramoleculeName: Mycobacterial open-gate proteasome capped with engineered bacterial enhancer-binding protein NtrC1 in presence of ADP-AlFx
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: NtrC1-GS4

MacromoleculeName: NtrC1-GS4 / type: protein_or_peptide / ID: 1
Details: AAA+ domain of NtrC1 (residues 121-387) carrying the mycobacterial proteasome interaction motif (LGQYL) at the C-terminus including a Gly-Ser linker of 4 residues
Enantiomer: LEVO
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLRKENELLR REKDLKEEEY VFESPKMKEI LEKIKKISCA ECPVLITGES GVGKEVVAR LIHKLSDRSK EPFVALNVAS IPRDIFEAEL FGYEKGAFTG A VSSKEGFF ELADGGTLFL DEIGELSLEA QAKLLRVIES GKFYRLGGRK EI EVNVRIL AATNRNIKEL ...String:
MLRKENELLR REKDLKEEEY VFESPKMKEI LEKIKKISCA ECPVLITGES GVGKEVVAR LIHKLSDRSK EPFVALNVAS IPRDIFEAEL FGYEKGAFTG A VSSKEGFF ELADGGTLFL DEIGELSLEA QAKLLRVIES GKFYRLGGRK EI EVNVRIL AATNRNIKEL VKEGKFREDL YYRLGVIEIE IPPLRERKED IIP LANHFL KKFSRKYAKE VEGFTKSAQE LLLSYPWYGN VRELKNVIER AVLF SEGKF IDRGELSCLV NSKGSGSLGQ YL

UniProtKB: Transcriptional regulator (NtrC family)

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Macromolecule #2: mycobacterial proteasome subunit alpha

MacromoleculeName: mycobacterial proteasome subunit alpha / type: protein_or_peptide / ID: 2
Details: deletion of 7 residues at the N-terminus (open-gate variant of the proteasome)
Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSPEQAMRER SELARKGIAR AKSVVALAYA GGVLFVAENP SRSLQKISEL YDRVGFAAAG KFNEFDNLRR GGIQFADTRG YAYDRRDVTG RQLANVYAQT LGTIFTEQAK PYEVELCVAE VAHYGETKRP ELYRITYDGS IADEPHFVVM GGTTEPIANA LKESYAENAS ...String:
MSPEQAMRER SELARKGIAR AKSVVALAYA GGVLFVAENP SRSLQKISEL YDRVGFAAAG KFNEFDNLRR GGIQFADTRG YAYDRRDVTG RQLANVYAQT LGTIFTEQAK PYEVELCVAE VAHYGETKRP ELYRITYDGS IADEPHFVVM GGTTEPIANA LKESYAENAS LTDALRIAVA ALRAGSADTS GGDQPTLGVA SLEVAVLDAN RPRRAFRRIT GSALQALLVD QESPQSDGES SG

UniProtKB: Proteasome subunit alpha

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Macromolecule #3: mycobacterial proteasome subunit beta

MacromoleculeName: mycobacterial proteasome subunit beta / type: protein_or_peptide / ID: 3
Details: deletion of N-terminal propreptide and addition of C-terminal Strep-tag
Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTTIVALKYP GGVVMAGDRR STQGNMISGR DVRKVYITDD YTATGIAGTA AVAVEFARLY AVELEHYEKL EGVPLTFAGK INRLAIMVRG NLAAAMQGLL ALPLLAGYDI HASDPQSAGR IVSFDAAGGW NIEEEGYQAV GSGSLFAKSS MKKLYSQVTD GDSGLRVAVE ...String:
MTTIVALKYP GGVVMAGDRR STQGNMISGR DVRKVYITDD YTATGIAGTA AVAVEFARLY AVELEHYEKL EGVPLTFAGK INRLAIMVRG NLAAAMQGLL ALPLLAGYDI HASDPQSAGR IVSFDAAGGW NIEEEGYQAV GSGSLFAKSS MKKLYSQVTD GDSGLRVAVE ALYDAADDDS ATGGPDLVRG IFPTAVIIDA DGAVDVPESR IAELARAIIE SRSGADTFGS DGGEKWSHPQ FEK

UniProtKB: Proteasome subunit beta

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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: 8
Details: 40 mM Tris-HCl, pH 8/RT, 200 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.5 mM ADP, 4 mM NaF, 1 mM AlCl3
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa / Details: 10 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average electron dose: 49.08 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 43660
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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