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- PDB-5upa: CryoEM structure of crosslinked E.coli RNA polymerase elongation ... -

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Entry
Database: PDB / ID: 5upa
TitleCryoEM structure of crosslinked E.coli RNA polymerase elongation complex
Descriptor(DNA-directed RNA polymerase subunit ...) x 4
Keywordstranscription/dna/rna / DNA-dependent RNA polymerase / bacterial transcription / ternary elongation complex / transcription arrest / transcription-dna-rna complex
Specimen sourceEscherichia coli o157:h7 / bacteria / image: Escherichia coli
Escherichia coli o45:k1 (strain s88 / expec) / bacteria / image: Escherichia coli
T7likevirus / virus
MethodElectron microscopy (4.05 A resolution / Single particle)
AuthorsKang, J.Y. / Darst, S.A.
CitationElife, 2017, 6

Elife, 2017, 6 StrPapers
Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex.
Jin Young Kang / Paul Dominic B Olinares / James Chen / Elizabeth A Campbell / Arkady Mustaev / Brian T Chait / Max E Gottesman / Seth A Darst

DateDeposition: Feb 2, 2017 / Release: Apr 5, 2017

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Assembly

Deposited unit
A: DNA (29-MER)
B: DNA (29-MER)
R: RNA (5'-R(*GP*CP*A*UP*UP*CP*AP*AP*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
hetero molecules


Theoretical massNumber of molelcules
Total (without water)392,52011
Polyers392,3648
Non-polymers1553
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (A2)41030
ΔGint (kcal/M)-186
Surface area (A2)144230
MethodPISA

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chainDNA (29-MER)


Mass: 8840.689 Da / Num. of mol.: 1 / Source: (synth.) T7likevirus
#2: DNA chainDNA (29-MER)


Mass: 8813.646 Da / Num. of mol.: 1 / Details: Template DNA / Source: (synth.) T7likevirus

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RNA chain , 1 types, 1 molecules R

#3: RNA chainRNA (5'-R(*GP*CP*A*UP*UP*CP*AP*AP*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')


Mass: 6509.968 Da / Num. of mol.: 1 / Source: (synth.) T7likevirus

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

#4: Polypeptide(L)DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 26459.125 Da / Num. of mol.: 2 / Source: (gene. exp.) Escherichia coli O157:H7 / References: UniProt: P0A7Z6, EC: 2.7.7.6
#5: Polypeptide(L)DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli O45:K1 (strain S88 / ExPEC)
References: UniProt: B7MIX3, EC: 2.7.7.6
#6: Polypeptide(L)DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli O157:H7 / References: UniProt: P0A8T8, EC: 2.7.7.6
#7: Polypeptide(L)DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 9094.239 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli O45:K1 (strain S88 / ExPEC)
References: UniProt: B7MFL0, EC: 2.7.7.6

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Non-polymers , 2 types, 3 molecules

#8: ChemicalChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg
#9: ChemicalChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentReconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Crosslinked E.coli RNA polymerase ternary elongation complex
Type: COMPLEX
Specimen supportGrid material: GOLD / Grid type: C-flat CF-1.2/1.3-3Au
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100

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Electron microscopy imaging

MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

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Processing

SoftwareName: PHENIX / Version: 1.10_2155: / Classification: refinement
ComputingStructure refinement: PHENIX (1.10_2155: phenix.real_space_refine)
3D reconstructionResolution: 4.05 A / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 83800 / Number of class averages: 1
Atomic model buildingRef space: REAL
Least-squares processHighest resolution: 4.05 A
Refine LS restraints
Refine idTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00727067
ELECTRON MICROSCOPYf_angle_d0.89236884
ELECTRON MICROSCOPYf_dihedral_angle_d9.56522392
ELECTRON MICROSCOPYf_chiral_restr0.0554221
ELECTRON MICROSCOPYf_plane_restr0.0054574

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