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- PDB-10mk: SemiClosed Mtb-EC: Cryo-EM structure of Mtb RNAP elongation compl... -

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Basic information

Entry
Database: PDB / ID: 10mk
TitleSemiClosed Mtb-EC: Cryo-EM structure of Mtb RNAP elongation complex (substrate loading mimic) with a semiclosed active site (closed TL, open RH-FL)
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-template DNA
  • RNA
  • Template DNA
KeywordsTRANSCRIPTION / RNA polymerase / DNA/RNA / Nucleotide addition cycle
Function / homology
Function and homology information


Antimicrobial action and antimicrobial resistance in Mtb / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity ...Antimicrobial action and antimicrobial resistance in Mtb / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / response to antibiotic / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytoplasm / cytosol
Similarity search - Function
DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime ...DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDhingra, Y. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118130 United States
CitationJournal: bioRxiv / Year: 2026
Title: RNA polymerase inhibitors reveal active-site motions essential for the nucleotide-addition cycle.
Authors: Yukti Dhingra / Robert Landick / Elizabeth A Campbell / Seth A Darst /
Abstract: The nucleotide-addition cycle (NAC) of multi-subunit DNA-dependent RNA polymerases (RNAPs) involves coordinated conformational changes in conserved active-site structural elements, including the ...The nucleotide-addition cycle (NAC) of multi-subunit DNA-dependent RNA polymerases (RNAPs) involves coordinated conformational changes in conserved active-site structural elements, including the trigger loop (TL). The TL is open (unfolded) in most RNAP structures but can close (fold) in substrate-bound (post- or pre-translocated) states of the RNAP, promoting catalysis. TL closure has been associated with closure of another conserved structural element, the Rim-Helices/F-loop (RH-FL), but the role of the RH-FL in the NAC is unclear. Antibiotic leads CBR9379 and AAP-SO inhibit the and RNAPs, respectively, by binding in a pocket formed by the bridge helix and RH-FL. The precise mechanism of action for these inhibitors is yet to be defined. We present cryo-electron microscopy structures showing that both compounds inhibit the RNAP NAC by preventing RH-FL closure, thereby allosterically destabilizing the closed TL. This work reveals a conserved mechanistic principle of RNAP catalysis across all domains of life and provides new insight for antibiotic design.
History
DepositionJan 27, 2026Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 24, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
E: DNA-directed RNA polymerase subunit omega
P: Template DNA
R: RNA
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
Q: Non-template DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)404,78213
Polymers404,0808
Non-polymers7035
Water16,286904
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABECD

#1: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 37745.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoA, Rv3457c, MTCY13E12.10c / Production host: Escherichia coli (E. coli) / References: UniProt: P9WGZ1, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11851.140 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoZ, Rv1390, MTCY21B4.07 / Production host: Escherichia coli (E. coli) / References: UniProt: P9WGY5, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 130018.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB, Rv0667, MTCI376.08c / Production host: Escherichia coli (E. coli) / References: UniProt: P9WGY9, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147097.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoC, Rv0668, MTCI376.07c / Production host: Escherichia coli (E. coli) / References: UniProt: P9WGY7, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules PQ

#3: DNA chain Template DNA


Mass: 16506.547 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#7: DNA chain Non-template DNA


Mass: 16589.572 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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RNA chain , 1 types, 1 molecules R

#4: RNA chain RNA


Mass: 6525.968 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 4 types, 909 molecules

#8: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 904 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mtb RNAP elongation complex with a substrate loading mimic
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX2.0_5885model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54790 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 64.34 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006124653
ELECTRON MICROSCOPYf_angle_d0.587933701
ELECTRON MICROSCOPYf_chiral_restr0.04573842
ELECTRON MICROSCOPYf_plane_restr0.00524192
ELECTRON MICROSCOPYf_dihedral_angle_d14.45719533

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