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- PDB-4v1m: Architecture of the RNA polymerase II-Mediator core transcription... -

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Entry
Database: PDB / ID: 4v1m
TitleArchitecture of the RNA polymerase II-Mediator core transcription initiation complex
Components
  • (DNA-DIRECTED RNA POLYMERASE II SUBUNIT ...Polymerase) x 5
  • (DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC ...RNA polymerase) x 5
  • 5'-D(*AP*AP*GP*TP*AP*CP*TP*TP*GP*AP)-3'
  • 5'-D(*CP*CP*AP*GP*GP*AP)-3'
  • 5'-D(*TP*CP*AP*AP*GP*TP*AP*CP*TP*TP*TP*TP*TP*CP *CP*BRUP*GP*GP*TP*C)-3'
KeywordsTRANSCRIPTION / TRANSCRIPTION INITIATION / RNA POLYMERASE II / GENERAL TRANSCRIPTION FACTORS
Function / homology
Function and homology information


RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / : / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / termination of RNA polymerase II transcription / Formation of TC-NER Pre-Incision Complex / RNA polymerase II transcribes snRNA genes ...RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / : / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / termination of RNA polymerase II transcription / Formation of TC-NER Pre-Incision Complex / RNA polymerase II transcribes snRNA genes / RNA Polymerase I Promoter Escape / TP53 Regulates Transcription of DNA Repair Genes / Estrogen-dependent gene expression / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / RNA-templated transcription / termination of RNA polymerase III transcription / RNA Polymerase II Pre-transcription Events / Dual incision in TC-NER / RNA polymerase I activity / termination of RNA polymerase I transcription / transcription initiation at RNA polymerase III promoter / tRNA transcription by RNA polymerase III / nucleolar large rRNA transcription by RNA polymerase I / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription initiation at RNA polymerase I promoter / RNA polymerase II activity / transcription elongation by RNA polymerase I / transcription-coupled nucleotide-excision repair / translesion synthesis / transcription by RNA polymerase I / RNA polymerase I complex / RNA polymerase III complex / transcription by RNA polymerase III / RNA polymerase II, core complex / transcription elongation by RNA polymerase II / transcription initiation at RNA polymerase II promoter / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / peroxisome / cytoplasmic stress granule / ribosome biogenesis / transcription by RNA polymerase II / nucleic acid binding / protein dimerization activity / mRNA binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 ...RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Zinc finger TFIIS-type signature. / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA-directed RNA polymerase subunit Rpo11 / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / RNA polymerases L / 13 to 16 Kd subunits signature. / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase II subunit RPB1 / DNA-directed RNA polymerase II subunit RPB2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 ...DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase II subunit RPB1 / DNA-directed RNA polymerase II subunit RPB2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB9 / DNA-directed RNA polymerase II subunit RPB11 / DNA-directed RNA polymerases I, II, and III subunit RPABC4
Similarity search - Component
Biological speciesSACCHAROMYCES CEREVISIAE (brewer's yeast)
SYNTHETIC CONSTRUCT (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsPlaschka, C. / Lariviere, L. / Wenzeck, L. / Hemann, M. / Tegunov, D. / Petrotchenko, E.V. / Borchers, C.H. / Baumeister, W. / Herzog, F. / Villa, E. / Cramer, P.
CitationJournal: Nature / Year: 2015
Title: Architecture of the RNA polymerase II-Mediator core initiation complex.
Authors: C Plaschka / L Larivière / L Wenzeck / M Seizl / M Hemann / D Tegunov / E V Petrotchenko / C H Borchers / W Baumeister / F Herzog / E Villa / P Cramer /
Abstract: The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all ...The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.
History
DepositionSep 29, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 4, 2015Provider: repository / Type: Initial release
Revision 1.1Feb 18, 2015Group: Database references
Revision 1.2Feb 25, 2015Group: Database references
Revision 1.3Sep 16, 2015Group: Other
Revision 2.0Aug 2, 2017Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Refinement description
Category: atom_site / em_3d_fitting ...atom_site / em_3d_fitting / em_image_scans / em_software / struct_conn
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.pdbx_formal_charge / _em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Assembly

Deposited unit
A: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1
B: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2
C: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3
E: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 1
F: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 2
H: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 3
I: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB9
J: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 5
K: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB11
L: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 4
N: 5'-D(*AP*AP*GP*TP*AP*CP*TP*TP*GP*AP)-3'
P: 5'-D(*CP*CP*AP*GP*GP*AP)-3'
T: 5'-D(*TP*CP*AP*AP*GP*TP*AP*CP*TP*TP*TP*TP*TP*CP *CP*BRUP*GP*GP*TP*C)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)481,28722
Polymers480,73913
Non-polymers5489
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA-DIRECTED RNA POLYMERASE II SUBUNIT ... , 5 types, 5 molecules ABCIK

#1: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1 / Polymerase / RNA POLYMERASE II SUBUNIT 1 / RNA POLYMERASE II SUBUNIT B1 / DNA-DIRECTED RNA POLYMERASE III ...RNA POLYMERASE II SUBUNIT 1 / RNA POLYMERASE II SUBUNIT B1 / DNA-DIRECTED RNA POLYMERASE III LARGEST SUBUNIT / RNA POLYMERASE II SUBUNIT B220


Mass: 191821.578 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P04050, DNA-directed RNA polymerase
#2: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2 / Polymerase / RNA POLYMERASE II SUBUNIT 2 / B150 / DNA-DIRECTED RNA POLYMERASE II 140 KDA POLYPEPTIDE


Mass: 138937.297 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P08518, DNA-directed RNA polymerase
#3: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3 / Polymerase / RNA POLYMERASE II SUBUNIT 3 / RNA POLYMERASE II SUBUNIT B3 / B44.5 / DNA-DIRECTED RNA POLYMERASE II ...RNA POLYMERASE II SUBUNIT 3 / RNA POLYMERASE II SUBUNIT B3 / B44.5 / DNA-DIRECTED RNA POLYMERASE II 45 KDA POLYPEPTIDE


Mass: 35330.457 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P16370
#7: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB9 / Polymerase / RNA POLYMERASE II SUBUNIT B9 / B12.6 / DNA-DIRECTED POLYMERASE II 14.2 KDA POLYPEPTIDE / DNA- ...RNA POLYMERASE II SUBUNIT B9 / B12.6 / DNA-DIRECTED POLYMERASE II 14.2 KDA POLYPEPTIDE / DNA-DIRECTED RNA POLYMERASE II SUBUNIT 9


Mass: 14308.161 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P27999
#9: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB11 / Polymerase / RNA POLYMERASE II SUBUNIT B11 / B13.6 / DNA-DIRECTED RNA POLYMERASE II 13.6 KDA POLYPEPTIDE


Mass: 13633.493 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P38902

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DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC ... , 5 types, 5 molecules EFHJL

#4: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 1 / RNA polymerase / RNA POLYMERASES I\ / II\ / AND III SUBUNIT ABC1 / ABC27 / DNA-DIRECTED RNA POLYMERASES I\ / II\ / ...RNA POLYMERASES I\ / II\ / AND III SUBUNIT ABC1 / ABC27 / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III 27 KDA POLYPEPTIDE / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III SUBUNIT RPABC 1


Mass: 25117.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P20434
#5: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 2 / RNA polymerase / RNA POLYMERASES I\ / II\ / AND III SUBUNIT ABC2 / ABC23 / DNA-DIRECTED RNA POLYMERASES I\ / II\ / ...RNA POLYMERASES I\ / II\ / AND III SUBUNIT ABC2 / ABC23 / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III 23 KDA POLYPEPTIDE / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III SUBUNIT RPABC 2


Mass: 17931.834 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P20435
#6: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 3 / RNA polymerase / RNA POLYMERASES I / II / AND III SUBUNIT ABC3 / ABC14.4 / ABC14.5 / DNA-DIRECTED RNA POLYMERASES I\ ...RNA POLYMERASES I / II / AND III SUBUNIT ABC3 / ABC14.4 / ABC14.5 / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III 14.5 KDA POLYPEPTIDE / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III SUBUNIT RPABC 3


Mass: 16525.363 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P20436
#8: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 5 / RNA polymerase / RNA POLYMERASES I\ / II\ / AND III SUBUNIT ABC5 / ABC10-BETA / ABC8 / DNA-DIRECTED RNA POLYMERASES ...RNA POLYMERASES I\ / II\ / AND III SUBUNIT ABC5 / ABC10-BETA / ABC8 / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III 8.3 KDA POLYPEPTIDE / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III SUBUNIT RPABC 5


Mass: 8290.732 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P22139
#10: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 4 / RNA polymerase / RNA POLYMERASES I\ / II\ / AND III SUBUNIT ABC4 / ABC10-ALPHA / DNA-DIRECTED RNA POLYMERASES I\ / ...RNA POLYMERASES I\ / II\ / AND III SUBUNIT ABC4 / ABC10-ALPHA / DNA-DIRECTED RNA POLYMERASES I\ / II\ / AND III SUBUNIT RPABC 4


Mass: 7729.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SACCHAROMYCES CEREVISIAE (brewer's yeast) / Strain: BJ5464 RPB3 HIS-BIO / References: UniProt: P40422, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#11: DNA chain 5'-D(*AP*AP*GP*TP*AP*CP*TP*TP*GP*AP)-3' / NONTEMPLATE DNA


Mass: 3068.042 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#13: DNA chain 5'-D(*TP*CP*AP*AP*GP*TP*AP*CP*TP*TP*TP*TP*TP*CP *CP*BRUP*GP*GP*TP*C)-3' / TEMPLATE DNA


Mass: 6130.794 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

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RNA chain , 1 types, 1 molecules P

#12: RNA chain 5'-D(*CP*CP*AP*GP*GP*AP)-3'


Mass: 1914.229 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

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Non-polymers , 2 types, 9 molecules

#14: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn
#15: Chemical ChemComp-MG / MAGNESIUM ION


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: POL II-DNA-RNA / Type: COMPLEX
Buffer solutionName: 25 MM HEPES-KOH PH 7.5, 180 MM POTASSIUM ACETATE, 5 % GLYCEROL, 5 MM DTT
pH: 7.5
Details: 25 MM HEPES-KOH PH 7.5, 180 MM POTASSIUM ACETATE, 5 % GLYCEROL, 5 MM DTT
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: OTHER
VitrificationCryogen name: ETHANE
Details: GRIDS WERE GLOW- DISCHARGED FOR 20 S BEFORE DEPOSITION OF 4 MICROLITERS SAMPLE AND INCUBATED FOR 30 S. GRIDS WERE WASHED TWICE WITH 4 MICROLITERS DISTILLED WATER, BLOTTED, AND VITRIFIED BY ...Details: GRIDS WERE GLOW- DISCHARGED FOR 20 S BEFORE DEPOSITION OF 4 MICROLITERS SAMPLE AND INCUBATED FOR 30 S. GRIDS WERE WASHED TWICE WITH 4 MICROLITERS DISTILLED WATER, BLOTTED, AND VITRIFIED BY PLUNGING INTO LIQUID ETHANE WITH A MANUAL PLUNGER.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Aug 1, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000 X / Calibrated magnification: 37169 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2 mm
Image recordingElectron dose: 25 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Situsmodel fitting
2RELION1.23D reconstruction
CTF correctionDetails: EACH PARTICLE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.6 Å / Num. of particles: 14777 / Nominal pixel size: 1.35 Å / Actual pixel size: 1.35 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2784. (DEPOSITION ID: 12825).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY
Atomic model buildingPDB-ID: 4A3D
RefinementHighest resolution: 6.6 Å
Refinement stepCycle: LAST / Highest resolution: 6.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms28697 741 9 0 29447

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