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- EMDB-48574: Mycobacterial open-gate proteasome in complex with engineered NtrC1 -
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Open data
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Basic information
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Title | Mycobacterial open-gate proteasome in complex with engineered NtrC1 | |||||||||
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![]() | sigma N / sigma 54 / ATPase / bacterial enhancer binding protein / transcription initiation / intermediate / TRANSCRIPTION | |||||||||
Function / homology | ![]() symbiont-mediated perturbation of host defenses / zymogen binding / DNA-binding transcription activator activity / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / phosphorelay signal transduction system / cis-regulatory region sequence-specific DNA binding ...symbiont-mediated perturbation of host defenses / zymogen binding / DNA-binding transcription activator activity / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / phosphorelay signal transduction system / cis-regulatory region sequence-specific DNA binding / proteolysis involved in protein catabolic process / peptidoglycan-based cell wall / modification-dependent protein catabolic process / protein-DNA complex / proteasome-mediated ubiquitin-dependent protein catabolic process / positive regulation of DNA-templated transcription / ATP hydrolysis activity / extracellular region / ATP binding / metal ion binding / identical protein binding / plasma membrane / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.6 Å | |||||||||
![]() | Molina N / Mueller AU / Darst SA | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 45.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 19.9 KB 19.9 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.4 KB | Display | ![]() |
Images | ![]() | 75.7 KB | ||
Masks | ![]() | 91.1 MB | ![]() | |
Filedesc metadata | ![]() | 5.7 KB | ||
Others | ![]() ![]() ![]() | 86 MB 84.5 MB 84.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.1 MB | Display | |
Data in XML | ![]() | 17.4 KB | Display | |
Data in CIF | ![]() | 22.8 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9mseC ![]() 9msfC ![]() 9msgC ![]() 9mshC ![]() 9msjC C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | unsharpened map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.5 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
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-Additional map: sharpened map (b-factor 103.3)
File | emd_48574_additional_1.map | ||||||||||||
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Annotation | sharpened map (b-factor 103.3) | ||||||||||||
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Density Histograms |
-Half map: #1
File | emd_48574_half_map_1.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_48574_half_map_2.map | ||||||||||||
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Sample components
-Entire : Mycobacterial open-gate proteasome capped with engineered bacteri...
Entire | Name: Mycobacterial open-gate proteasome capped with engineered bacterial enhancer-binding protein NtrC1 in presence of ADP-AlFx |
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Components |
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-Supramolecule #1: Mycobacterial open-gate proteasome capped with engineered bacteri...
Supramolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: NtrC1-GS4
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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) |
-Macromolecule #2: mycobacterial proteasome subunit alpha
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #3: mycobacterial proteasome subunit beta
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 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 |
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Grid | Model: 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 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film 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 beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination 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 |