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- PDB-5oik: Structure of an RNA polymerase II-DSIF transcription elongation c... -

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Entry
Database: PDB / ID: 5oik
TitleStructure of an RNA polymerase II-DSIF transcription elongation complex
DescriptorTranscription elongation factor SPT5
KeywordsTRANSCRIPTION / RNA polymerase II / transcription elongation
Specimen sourceHomo sapiens / human
Bos taurus / mammal / Bovine / ウシ /
Synthetic construct
MethodElectron microscopy (3.7 Å resolution / Particle / Single particle)
AuthorsBernecky, C. / Plitzko, J.M. / Cramer, P.
CitationNat. Struct. Mol. Biol., 2017

Nat. Struct. Mol. Biol., 2017 StrPapers
Structure of a transcribing RNA polymerase II-DSIF complex reveals a multidentate DNA-RNA clamp.
Carrie Bernecky / Jürgen M Plitzko / Patrick Cramer

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 18, 2017 / Release: Sep 13, 2017

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Assembly

Deposited unit
A: DNA-directed RNA polymerase II subunit RPB1
B: DNA-directed RNA polymerase subunit beta
C: DNA-directed RNA polymerase II subunit RPB3
D: Polymerase (RNA) II (DNA directed) polypeptide D
E: DNA-directed RNA polymerases I, II, and III subunit RPABC1
F: DNA-directed RNA polymerases I, II, and III subunit RPABC2
G: DNA-directed RNA polymerase II subunit RPB7
H: Uncharacterized protein
I: DNA-directed RNA polymerase II subunit RPB9
J: DNA-directed RNA polymerases I, II, and III subunit RPABC5
K: DNA-directed RNA polymerase II subunit RPB11
L: DNA-directed RNA polymerases I, II, and III subunit RPABC4
N: DNA (43-MER)
P: RNA (5'-R(P*UP*AP*UP*AP*UP*AP*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
T: DNA (43-MER)
Y: Transcription elongation factor SPT4
Z: Transcription elongation factor SPT5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)694,73026
Polyers694,18317
Non-polymers5489
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

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DNA-directed RNA polymerase II subunit ... , 5 types, 5 molecules ACGIK

#1: Polypeptide(L)DNA-directed RNA polymerase II subunit RPB1


Mass: 217436.062 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: EC: 2.7.7.6
#3: Polypeptide(L)DNA-directed RNA polymerase II subunit RPB3 / RNA polymerase II subunit B3 / DNA-directed RNA polymerase II subunit C


Mass: 31466.098 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: Q3T0Q3
#7: Polypeptide(L)DNA-directed RNA polymerase II subunit RPB7 / RNA polymerase II subunit B7 / DNA-directed RNA polymerase II subunit G


Mass: 19314.283 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: Q5E9B8
#9: Polypeptide(L)DNA-directed RNA polymerase II subunit RPB9 / RNA polymerase II subunit B9 / DNA-directed RNA polymerase II subunit I


Mass: 14507.205 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: Q32P73
#11: Polypeptide(L)DNA-directed RNA polymerase II subunit RPB11 / RNA polymerase II subunit B11 / DNA-directed RNA polymerase II subunit J


Mass: 13310.284 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: Q32P79

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Polypeptide(L) , 3 types, 3 molecules BDH

#2: Polypeptide(L)DNA-directed RNA polymerase subunit beta


Mass: 134041.422 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: A5PJW8, EC: 2.7.7.6
#4: Polypeptide(L)Polymerase (RNA) II (DNA directed) polypeptide D


Mass: 16347.255 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: Q1JQ91
#8: Polypeptide(L)Uncharacterized protein


Mass: 17162.273 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: F2Z4H3

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DNA-directed RNA polymerases I, II, and III subunit ... , 4 types, 4 molecules EFJL

#5: Polypeptide(L)DNA-directed RNA polymerases I, II, and III subunit RPABC1 / RNA polymerases I / II / and III subunit ABC1 / DNA-directed RNA polymerase II subunit E / RPB5 homolog


Mass: 24514.219 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: Q2T9T3
#6: Polypeptide(L)DNA-directed RNA polymerases I, II, and III subunit RPABC2


Mass: 14491.026 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: F2Z4C9
#10: Polypeptide(L)DNA-directed RNA polymerases I, II, and III subunit RPABC5 / RNA polymerases I / II / and III subunit ABC5 / DNA-directed RNA polymerase III subunit L


Mass: 7655.123 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: Q32P78
#12: Polypeptide(L)DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerases I / II / and III subunit ABC4 / DNA-directed RNA polymerase II subunit K


Mass: 7018.244 Da / Num. of mol.: 1 / Source: (natural) Bos taurus / References: UniProt: Q3ZBC0

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DNA chain , 2 types, 2 molecules NT

#13: DNA chainDNA (43-MER)


Mass: 13303.563 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct
#15: DNA chainDNA (43-MER)


Mass: 13178.483 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct

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RNA chain , 1 types, 1 molecules P

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


Mass: 16081.696 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct

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Transcription elongation factor ... , 2 types, 2 molecules YZ

#16: Polypeptide(L)Transcription elongation factor SPT4 / hSPT4 / DRB sensitivity-inducing factor 14 kDa subunit / DSIF p14 / DRB sensitivity-inducing factor small subunit / DSIF small subunit


Mass: 13210.201 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens / References: UniProt: P63272
#17: Polypeptide(L)Transcription elongation factor SPT5 / hSPT5 / DRB sensitivity-inducing factor 160 kDa subunit / DSIF p160 / DRB sensitivity-inducing factor large subunit / DSIF large subunit / Tat-cotransactivator 1 protein / Tat-CT1 protein


Mass: 121145.477 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens / References: UniProt: O00267

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Non-polymers , 2 types, 9 molecules

#18: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Formula: Zn
#19: ChemicalChemComp-MG / MAGNESIUM ION


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

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Details

Compound detailsIn order to illustrate the path of the nontemplate DNA, phosphate atoms (chain N residues 15-23) were placed in the center of the observed DNA density.
Sequence detailsChain A , corresponds to NCBI entry NP_001193242.1

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1RNA polymerase II-DSIF elongation complexCOMPLEX1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 170MULTIPLE SOURCES
2RNA polymerase IICOMPLEX1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 121NATURAL
3DSIFCOMPLEX16, 171RECOMBINANT
4DNA-RNA synthetic constructCOMPLEX13, 14, 151RECOMBINANT
Molecular weight
IDValueUnitsEntity assembly IDExperimental value
10.65MEGADALTONS1NO
20.52MEGADALTONS1NO
30.13MEGADALTONS1NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
119606Homo sapiens
229913Bos taurus
339606Homo sapiens
4432630synthetic construct
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismStrain
11469008Escherichia coliBL21(DE3)
23469008Escherichia coliBL21(DE3)
3432630synthetic construct
Buffer solutionDetails: pH was adjusted at 25 degrees Celsius / pH: 7.25
Buffer component
IDConc.UnitsNameFormulaBuffer ID
15mMHEPES1
2150mMsodium chlorideNaCl1
30.01mMzinc chlorideZnCl21
41mMdithiothreitol1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins
Details: Sample (four microliters) was applied to glow-discharged Quantifoil R 2/1 holey carbon grids, which were then blotted for 8.5s and plunge-frozen in liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 37000 / Calibrated magnification: 37037 / Nominal defocus max: 3600 nm / Nominal defocus min: 600 nm / Calibrated defocus min: 600 nm / Calibrated defocus max: 3600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9.9 sec. / Electron dose: 33 e/Å2
Details: Movies were aligned and binned to the physical pixel size of 1.35 angstroms.
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 2549
EM imaging opticsEnergyfilter name: GIF Quantum / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansMovie frames/image: 33 / Used frames/image: 1-33

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Processing

SoftwareName: PHENIX / Version: 1.10_2155: / Classification: refinement
EM software
IDNameVersionCategoryImage processing IDImaging IDFitting ID
1RELION1.4PARTICLE SELECTION1
2TOM ToolboxIMAGE ACQUISITION1
4CTFFIND4.0.16CTF CORRECTION1
7Rosetta3.6MODEL FITTING1
9RELION1.4INITIAL EULER ASSIGNMENT1
10RELION1.4FINAL EULER ASSIGNMENT1
12RELION1.4RECONSTRUCTION1
13PHENIX1.10MODEL REFINEMENT1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 687928
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 101140 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingRef space: REAL
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01138891
ELECTRON MICROSCOPYf_angle_d1.05952944
ELECTRON MICROSCOPYf_dihedral_angle_d10.91623475
ELECTRON MICROSCOPYf_chiral_restr0.0615931
ELECTRON MICROSCOPYf_plane_restr0.0066516

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