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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
Components
  • (DNA (43-MER)) x 2
  • (DNA-directed RNA polymerase II subunit ...) x 5
  • (DNA-directed RNA polymerases I, II, and III subunit ...) x 4
  • (Transcription elongation factor ...) x 2
  • DNA-directed RNA polymerase subunit beta
  • Polymerase (RNA) II (DNA directed) polypeptide D
  • 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')
  • Uncharacterized protein
KeywordsTRANSCRIPTION / RNA polymerase II / transcription elongation
Function / homology
Function and homology information


Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase II Promoter Escape ...Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase I Transcription Termination / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase III Transcription Initiation From Type 3 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Estrogen-dependent gene expression / RNA Polymerase II Pre-transcription Events / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase II Transcription Elongation / Processing of Capped Intron-Containing Pre-mRNA / B-WICH complex positively regulates rRNA expression / mRNA Splicing - Major Pathway / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / negative regulation of DNA-templated transcription, elongation / DNA/RNA hybrid binding / DSIF complex / regulation of transcription elongation by RNA polymerase II / positive regulation of DNA-templated transcription, elongation / Abortive elongation of HIV-1 transcript in the absence of Tat / transcription elongation-coupled chromatin remodeling / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / Formation of the HIV-1 Early Elongation Complex / mRNA Capping / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / RNA polymerase II complex binding / negative regulation of transcription elongation by RNA polymerase II / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / transcription by RNA polymerase I / positive regulation of macroautophagy / nuclear-transcribed mRNA catabolic process / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / transcription by RNA polymerase III / RNA polymerase II transcribes snRNA genes / RNA polymerase II activity / Tat-mediated elongation of the HIV-1 transcript / transcription elongation by RNA polymerase I / Formation of HIV-1 elongation complex containing HIV-1 Tat / transcription-coupled nucleotide-excision repair / tRNA transcription by RNA polymerase III / RNA polymerase I complex / RNA polymerase III complex / positive regulation of translational initiation / Formation of HIV elongation complex in the absence of HIV Tat / RNA polymerase II, core complex / RNA Polymerase II Transcription Elongation / core promoter sequence-specific DNA binding / Formation of RNA Pol II elongation complex / RNA Polymerase II Pre-transcription Events / translation initiation factor binding / protein-DNA complex / TP53 Regulates Transcription of DNA Repair Genes / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / P-body / euchromatin / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / single-stranded DNA binding / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / transcription by RNA polymerase II / nucleic acid binding / chromosome, telomeric region / single-stranded RNA binding / protein dimerization activity / hydrolase activity / protein heterodimerization activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / mRNA binding / nucleotide binding / chromatin binding / regulation of DNA-templated transcription / nucleolus / negative regulation of transcription by RNA polymerase II / enzyme binding / magnesium ion binding / positive regulation of transcription by RNA polymerase II / DNA binding / RNA binding
Similarity search - Function
Herpes Virus-1 - #210 / Spt5, KOW domain repeat 6 / RNA Polymerase II, Rpb4 subunit / RNA polymerase Rpb7-like, N-terminal domain / Transcription initiation Spt4 / Spt4 superfamily / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / Transcription elongation factor Spt5, eukaryote ...Herpes Virus-1 - #210 / Spt5, KOW domain repeat 6 / RNA Polymerase II, Rpb4 subunit / RNA polymerase Rpb7-like, N-terminal domain / Transcription initiation Spt4 / Spt4 superfamily / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / Transcription elongation factor Spt5, eukaryote / Spt5 transcription elongation factor, N-terminal / Spt5, KOW domain repeat 2 / Spt5, KOW domain repeat 3 / Spt5, KOW domain repeat 5 / Spt5 transcription elongation factor, acidic N-terminal / NGN domain, eukaryotic / Spt5, KOW domain repeat 1 / Spt5, KOW domain repeat 4 / Spt5 C-terminal domain / Spt5 C-terminal nonapeptide repeat binding Spt4 / NGN domain / Transcription elongation factor SPT5 / Early transcription elongation factor of RNA pol II, NGN section / Dna-directed Rna Polymerases I, Ii, And Iii 27 Kd Polypeptide; Chain: A; domain 1 / RNA polymerase, Rpb5, N-terminal domain / RNA polymerase ii, chain L / RPB5-like RNA polymerase subunit / N-terminal domain of TfIIb - #10 / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / NusG, N-terminal domain superfamily / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Growth Hormone; Chain: A; / N-terminal domain of TfIIb / Rubrerythrin, domain 2 / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / 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 / Gyrase A; domain 2 / Rpb4/RPC9 superfamily / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Herpes Virus-1 / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Zinc finger TFIIS-type signature. / HRDC-like superfamily / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / 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, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / : / Homeodomain-like / 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, 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, 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 Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / Zinc finger, TFIIS-type
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase II subunit RPB2 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit RPB1 / Transcription elongation factor SPT5 / Transcription elongation factor SPT4 ...DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase II subunit RPB2 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit RPB1 / Transcription elongation factor SPT5 / Transcription elongation factor SPT4 / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerase II subunit RPB9 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB11 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / DNA-directed RNA polymerase II subunit RPB7
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBernecky, C. / Plitzko, J.M. / Cramer, P.
Funding support Germany, 3items
OrganizationGrant numberCountry
German Research FoundationSFB860, SPP1935 Germany
European Research CouncilTRANSREGULON Germany
Volkswagen Foundation Germany
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Structure of a transcribing RNA polymerase II-DSIF complex reveals a multidentate DNA-RNA clamp.
Authors: Carrie Bernecky / Jürgen M Plitzko / Patrick Cramer /
Abstract: During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA ...During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA processing. We combined cryo-EM and X-ray crystallography to determine the structure of the mammalian Pol II-DSIF elongation complex at a nominal resolution of 3.4 Å. Human DSIF has a modular structure with two domains forming a DNA clamp, two domains forming an RNA clamp, and one domain buttressing the RNA clamp. The clamps maintain the transcription bubble, position upstream DNA, and retain the RNA transcript in the exit tunnel. The mobile C-terminal region of DSIF is located near exiting RNA, where it can recruit factors for RNA processing. The structure provides insight into the roles of DSIF during mRNA synthesis.
History
DepositionJul 18, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 13, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 24, 2018Group: Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name ..._entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name / _entity_src_gen.pdbx_host_org_strain / _entity_src_gen.pdbx_host_org_variant
Revision 1.3Nov 6, 2019Group: Data collection / Database references / Category: pdbx_database_related / Item: _pdbx_database_related.content_type
Revision 1.4Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5May 15, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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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
Polymers694,18317
Non-polymers5489
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, native gel electrophoresis, microscopy
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: Protein DNA-directed RNA polymerase II subunit RPB1


Mass: 217436.062 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle)
References: UniProt: G3MZY8*PLUS, DNA-directed RNA polymerase
#3: Protein 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 method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q3T0Q3
#7: Protein 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 method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q5E9B8
#9: Protein 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 method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q32P73
#11: Protein 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 method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q32P79

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Protein , 3 types, 3 molecules BDH

#2: Protein DNA-directed RNA polymerase subunit beta


Mass: 134041.422 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: A5PJW8, DNA-directed RNA polymerase
#4: Protein Polymerase (RNA) II (DNA directed) polypeptide D


Mass: 16347.255 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q1JQ91
#8: Protein Uncharacterized protein


Mass: 17162.273 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: F2Z4H3

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

#5: Protein 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 method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q2T9T3
#6: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC2


Mass: 14491.026 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: F2Z4C9, UniProt: Q32PE0*PLUS
#10: Protein 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 method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q32P78
#12: Protein 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 method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q3ZBC0

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DNA chain , 2 types, 2 molecules NT

#13: DNA chain DNA (43-MER)


Mass: 13303.563 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#15: DNA chain DNA (43-MER)


Mass: 13178.483 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

#14: RNA chain 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')


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

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

#16: Protein Transcription elongation factor SPT4 / hSPT4 / DRB sensitivity-inducing factor 14 kDa subunit / DSIF p14 / DRB sensitivity-inducing factor ...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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SUPT4H1, SPT4H, SUPT4H / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIL / References: UniProt: P63272
#17: Protein Transcription elongation factor SPT5 / hSPT5 / DRB sensitivity-inducing factor 160 kDa subunit / DSIF p160 / DRB sensitivity-inducing ...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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SUPT5H, SPT5, SPT5H / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIL / 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 / Source method: obtained synthetically / Formula: Zn
#19: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / 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) ...In 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 / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1RNA polymerase II-DSIF elongation complexCOMPLEX#1-#170MULTIPLE SOURCES
2RNA polymerase IICOMPLEX#1-#121NATURAL
3DSIFCOMPLEX#16-#171RECOMBINANT
4DNA-RNA synthetic constructCOMPLEX#13-#151RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
120.52 MDaNO
230.13 MDaNO
340.04 MDaNO
410.69 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Bos taurus (cattle)9913
33Homo sapiens (human)9606
44synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
23Escherichia coli (E. coli)469008BL21(DE3)
34synthetic construct (others)32630
Buffer solutionpH: 7.25 / Details: pH was adjusted at 25 degrees Celsius
Buffer component
IDConc.NameFormulaBuffer-ID
15 mMHEPES1
2150 mMsodium chlorideNaCl1
30.01 mMzinc chlorideZnCl21
41 mMdithiothreitol1
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 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 37000 X / Calibrated magnification: 37037 X / 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 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9.9 sec. / Electron dose: 33 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2549
Details: Movies were aligned and binned to the physical pixel size of 1.35 angstroms.
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
IDNameVersionCategory
1RELION1.4particle selection
2TOM Toolboximage acquisition
4CTFFIND4.0.16CTF correction
7Rosetta3.6model fitting
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
12RELION1.43D reconstruction
13PHENIX1.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 687928
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101140 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingSpace: 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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