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- PDB-6f6w: Structure of Mycobacterium smegmatis RNA polymerase core -

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Basic information

Entry
Database: PDB / ID: 6f6w
TitleStructure of Mycobacterium smegmatis RNA polymerase core
Components(DNA-directed RNA polymerase subunit ...Polymerase) x 4
KeywordsTRANSCRIPTION / TRANSCRIPTION Sigma
Function / homology
Function and homology information


ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / transcription, DNA-templated / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding
RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / DNA-directed RNA polymerase, alpha subunit / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, domain 2 ...RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / DNA-directed RNA polymerase, alpha subunit / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, domain 2 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, insert domain / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb1, domain 3 superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb2, domain 3 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase, alpha subunit, C-terminal / RNA polymerase Rpb2, domain 7 / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase, alpha subunit / DNA-directed RNA polymerase, omega subunit / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase, N-terminal / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 6 / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Gyrase A; domain 2 / Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.81 Å
AuthorsKouba, T. / Barvik, I. / Krasny, L.
CitationJournal: J Bacteriol / Year: 2019
Title: The Core and Holoenzyme Forms of RNA Polymerase from .
Authors: Tomáš Kouba / Jiří Pospíšil / Jarmila Hnilicová / Hana Šanderová / Ivan Barvík / Libor Krásný /
Abstract: Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported ...Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of RNAP: core and holoenzyme containing σ but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σ is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 6, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 19, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 6, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.title ..._citation.journal_volume / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jul 31, 2019Group: Data collection / Refinement description / Category: refine
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
hetero molecules


Theoretical massNumber of molelcules
Total (without water)364,8308
Polymers364,6745
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area31660 Å2
ΔGint-176 kcal/mol
Surface area112120 Å2
MethodPISA

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Components

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

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


Mass: 37959.441 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: rpoA, MSMEG_1524, MSMEI_1488 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QSL8, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 129456.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: rpoB, MSMEG_1367, MSMEI_1328 / Production host: Escherichia coli (E. coli) / References: UniProt: P60281, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147785.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: rpoC, MSMEG_1368, MSMEI_1329 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QS66, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11512.698 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: rpoZ, MSMEG_3053, MSMEI_2977 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QWT1, DNA-directed RNA polymerase

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Non-polymers , 2 types, 3 molecules

#5: Chemical ChemComp-ZN / ZINC ION / Zinc


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


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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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: Mycobacterium smegmatis RNA polymerase Sigma-A holoenzyme
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.364 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium smegmatis str. MC2 155 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 78000 X / Nominal defocus max: 3800 nm / Nominal defocus min: 1500 nm / Cs: 2 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 2 sec. / Electron dose: 52 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0238 / Classification: refinement
EM software
IDNameVersionCategory
6Coot0.8.3model fitting
8REFMAC5.8model refinement
12RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL
RefinementResolution: 3.8→278.72 Å / Cor.coef. Fo:Fc: 0.899 / SU B: 32.343 / SU ML: 0.433 / ESU R: 0.259
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.3989 --
Obs0.3989 826173 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 103.679 Å2
Baniso -1Baniso -2Baniso -3
1--1.52 Å2-8.95 Å2-3.4 Å2
2--7.15 Å24.02 Å2
3----5.63 Å2
Refinement stepCycle: 1 / Total: 21619
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.01321976
ELECTRON MICROSCOPYr_bond_other_d00.01720705
ELECTRON MICROSCOPYr_angle_refined_deg1.2771.64529843
ELECTRON MICROSCOPYr_angle_other_deg1.0631.57647786
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.1552849
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.00921.521125
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.33153647
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.95815187
ELECTRON MICROSCOPYr_chiral_restr0.0440.22954
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.0224911
ELECTRON MICROSCOPYr_gen_planes_other0.0020.024478
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it2.82211.511435
ELECTRON MICROSCOPYr_mcbond_other2.82211.511433
ELECTRON MICROSCOPYr_mcangle_it5.35217.22614271
ELECTRON MICROSCOPYr_mcangle_other5.35217.22614271
ELECTRON MICROSCOPYr_scbond_it1.31111.42810541
ELECTRON MICROSCOPYr_scbond_other1.31111.42810542
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other2.97817.08215573
ELECTRON MICROSCOPYr_long_range_B_refined10.21824844
ELECTRON MICROSCOPYr_long_range_B_other10.21924842
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.649 60808 -
Obs--100 %

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