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- PDB-6fed: Structure of Mycobacterium smegmatis RNA polymerase Sigma-A holoenzyme -

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Entry
Database: PDB / ID: 6fed
TitleStructure of Mycobacterium smegmatis RNA polymerase Sigma-A holoenzyme
Components(DNA-directed RNA polymerase subunit ...Polymerase) x 4
KeywordsTRANSCRIPTION / TRANSCRIPTION Sigma
Function / homologyRNA polymerase Rpb2, OB-fold / RNA polymerase Rpb1, funnel domain superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 5 / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain / RPB6/omega subunit-like superfamily / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily ...RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb1, funnel domain superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 5 / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain / RPB6/omega subunit-like superfamily / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb2, domain 6 / DNA-directed RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb6 / RNA polymerase Rpb3/Rpb11 dimerisation domain / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb1, domain 1 / DNA-directed RNA polymerase, insert domain / RNA polymerases beta chain signature. / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 4 / 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 / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, alpha subunit, C-terminal / RNA polymerase Rpb1, domain 3 / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / transcription, DNA-templated / response to antibiotic / DNA binding / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta
Function and homology information
Specimen sourceMycobacterium smegmatis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.81 Å resolution
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
DateDeposition: Jan 1, 2018 / Release: Jan 30, 2019

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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
Polyers364,6745
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)32780
ΔGint (kcal/M)-172
Surface area (Å2)116560
MethodPISA

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Components

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

#1: Protein/peptide 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: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Gene: rpoA, MSMEG_1524, MSMEI_1488 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QSL8, DNA-directed RNA polymerase
#2: Protein/peptide 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: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Gene: rpoB, MSMEG_1367, MSMEI_1328 / Production host: Escherichia coli (E. coli) / References: UniProt: P60281, DNA-directed RNA polymerase
#3: Protein/peptide 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: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Gene: rpoC, MSMEG_1368, MSMEI_1329 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QS66, DNA-directed RNA polymerase
#4: Protein/peptide 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: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)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


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / Zinc
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Mycobacterium smegmatis RNA polymerase core / Type: COMPLEX / Entity ID: 1, 2, 3, 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
MicroscopyMicroscope model: 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 / Nominal defocus max: 3800 nm / Nominal defocus min: 1500 nm / Cs: 2 mm / C2 aperture diameter: 50 microns / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 2 sec. / Electron dose: 52 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0191 / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
4GctfCTF correction
7Coot0.8.3model fitting
12RELION2.13D reconstruction
13REFMAC5.8model refinement
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.81 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 163000 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: RECIPROCAL
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcDetailsR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection obsPercent reflection obsOverall SU BOverall SU MLOverall ESU RStereochemistry target valuesSolvent model details
1136.532-1.88-8.94-3.397.553.69-5.670.684HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS0.435370.435373.80278.72826173100.0036.7870.4800.284MAXIMUM LIKELIHOOD WITH PHASESPARAMETERS FOR MASK CACLULATION
ELECTRON MICROSCOPY
Number of atoms included #1Total: 21841
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.01922202
ELECTRON MICROSCOPYr_bond_other_d0.0260.02020915
ELECTRON MICROSCOPYr_angle_refined_deg0.9831.97530147
ELECTRON MICROSCOPYr_angle_other_deg0.8713.00048271
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.8335.0002880
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.67723.808948
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.94515.0003693
ELECTRON MICROSCOPYr_dihedral_angle_4_deg10.97215.000190
ELECTRON MICROSCOPYr_chiral_restr0.0530.2003500
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.02124988
ELECTRON MICROSCOPYr_gen_planes_other0.0020.0204340
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_it2.77314.23711553
ELECTRON MICROSCOPYr_mcbond_other2.77314.23711552
ELECTRON MICROSCOPYr_mcangle_it5.32021.33914422
ELECTRON MICROSCOPYr_mcangle_other5.32021.33914423
ELECTRON MICROSCOPYr_scbond_it0.91814.14310649
ELECTRON MICROSCOPYr_scbond_other0.91814.14310650
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other2.44221.11815726
ELECTRON MICROSCOPYr_long_range_B_refined9.84724505
ELECTRON MICROSCOPYr_long_range_B_other9.84724505
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS shellHighest resolution: 3.8 Å / R factor R work: 0.679 / Lowest resolution: 3.899 Å / Number reflection R free: 0 / Number reflection R work: 60808 / Total number of bins used: 20 / Percent reflection obs: 1

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