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- PDB-5w65: RNA polymerase I Initial Transcribing Complex State 2 -

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Basic information

Entry
Database: PDB / ID: 5w65
TitleRNA polymerase I Initial Transcribing Complex State 2
Components
  • (DNA-directed RNA polymerase I subunit ...) x 7
  • (DNA-directed RNA polymerases I and III subunit ...) x 2
  • (DNA-directed RNA polymerases I, II, and III subunit ...) x 5
  • (RNA polymerase I-specific transcription initiation factor ...) x 3
  • RNA
  • non-template strand DNA
  • template strand DNA
KeywordsTRANSCRIPTION / RNA Polymerase I Core Factor Transcription
Function / homology
Function and homology information


RNA polymerase I transcription regulatory region sequence-specific DNA binding / RNA polymerase I core factor complex / RNA polymerase I general transcription initiation factor activity / RNA polymerase I general transcription initiation factor binding / RNA polymerase I core promoter sequence-specific DNA binding / RNA polymerase I preinitiation complex assembly / RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase III Transcription Initiation From Type 2 Promoter / regulation of cell size ...RNA polymerase I transcription regulatory region sequence-specific DNA binding / RNA polymerase I core factor complex / RNA polymerase I general transcription initiation factor activity / RNA polymerase I general transcription initiation factor binding / RNA polymerase I core promoter sequence-specific DNA binding / RNA polymerase I preinitiation complex assembly / RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase III Transcription Initiation From Type 2 Promoter / regulation of cell size / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes / 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 / RNA Polymerase II Pre-transcription Events / RNA-templated transcription / termination of RNA polymerase III transcription / Formation of TC-NER Pre-Incision Complex / transcription initiation at RNA polymerase III promoter / termination of RNA polymerase I transcription / RNA polymerase III activity / RNA Polymerase I Promoter Escape / nucleolar large rRNA transcription by RNA polymerase I / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription by RNA polymerase I / transcription initiation at RNA polymerase I promoter / Estrogen-dependent gene expression / transcription by RNA polymerase III / Dual incision in TC-NER / transcription elongation by RNA polymerase I / tRNA transcription by RNA polymerase III / RNA polymerase I complex / RNA polymerase I activity / RNA polymerase III complex / RNA polymerase II, core complex / TBP-class protein binding / promoter-specific chromatin binding / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / ribonucleoside binding / DNA-directed RNA polymerase / peroxisome / ribosome biogenesis / RNA polymerase II-specific DNA-binding transcription factor binding / transcription by RNA polymerase II / nucleic acid binding / protein dimerization activity / nucleolus / negative regulation of transcription by RNA polymerase II / DNA binding / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytoplasm
Similarity search - Function
Transcription initiation factor Rrn11 / RNA polymerase I-specific transcription initiation factor Rrn6 / Transcription initiation factor Rrn11, budding yeast / RNA polymerase I-specific transcription initiation factor RRN6-like / : / : / : / : / RNA polymerase I-specific transcription initiation factor Rrn11 / Rrn6, beta-propeller ...Transcription initiation factor Rrn11 / RNA polymerase I-specific transcription initiation factor Rrn6 / Transcription initiation factor Rrn11, budding yeast / RNA polymerase I-specific transcription initiation factor RRN6-like / : / : / : / : / RNA polymerase I-specific transcription initiation factor Rrn11 / Rrn6, beta-propeller / RRN6, K-rich C-terminal domain / Rrn6, helical bundle domain / Transcription initiation factor Rrn7, Zinc-finger / RNA polymerase I transcription initiation factor TAF1B/Rrn7 / : / : / Zinc-finger of RNA-polymerase I-specific TFIIB, Rrn7 / Rrn7/TAF1B, N-terminal cyclin domain / Rrn7/TAF1B, C-terminal cyclin domain / : / RNA polymerase I, subunit Rpa14, fungi / Yeast RNA polymerase I subunit RPA14 / DNA-directed RNA polymerase I, subunit RPA34.5 / DNA-directed RNA polymerase I subunit RPA34.5 / RNA polymerase I associated factor, A49-like / A49-like RNA polymerase I associated factor / Rpa43, N-terminal ribonucleoprotein (RNP) domain / RPA43, OB domain / RPA43 OB domain in RNA Pol I / DNA-directed RNA polymerase I subunit RPA2, domain 4 / DNA-directed RNA pol I, largest subunit / Pol I subunit A12, C-terminal zinc ribbon / : / RNA polymerase I, Rpa2 specific domain / DNA-directed RNA polymerases I and III subunit AC19 / DNA-directed RNA polymerases I and III subunit AC40 / Zinc finger TFIIS-type signature. / 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 / 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 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, 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 / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / 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. / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / DNA-directed RNA polymerases I and III subunit RPAC1 / DNA-directed RNA polymerase I subunit RPA190 / 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 polymerase I subunit RPA135 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 ...DNA / DNA (> 10) / RNA / DNA-directed RNA polymerases I and III subunit RPAC1 / DNA-directed RNA polymerase I subunit RPA190 / 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 polymerase I subunit RPA135 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerases I and III subunit RPAC2 / DNA-directed RNA polymerase I subunit RPA12 / RNA polymerase I-specific transcription initiation factor RRN6 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase I-specific transcription initiation factor RRN7 / DNA-directed RNA polymerase I subunit RPA43 / DNA-directed RNA polymerase I subunit RPA34 / DNA-directed RNA polymerase I subunit RPA14 / DNA-directed RNA polymerase I subunit RPA49 / RNA polymerase I-specific transcription initiation factor RRN11
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Saccharomyces cerevisiae S288c (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsHan, Y. / He, Y.
Funding support United States, 5items
OrganizationGrant numberCountry
Northwestern UniversityCornew Innovation Award United States
American Cancer SocietyIRG-15-173-21 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)NCI 5K22CA184235 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIGMS GM110387 United States
National Science Foundation (NSF, United States)MCB-1149521 United States
CitationJournal: Elife / Year: 2017
Title: Structural mechanism of ATP-independent transcription initiation by RNA polymerase I.
Authors: Yan Han / Chunli Yan / Thi Hoang Duong Nguyen / Ashleigh J Jackobel / Ivaylo Ivanov / Bruce A Knutson / Yuan He /
Abstract: Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a ...Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a structure of Pol I-CF-DNA to 3.8 Å resolution using single-particle cryo-electron microscopy. The structure reveals a bipartite architecture of Core Factor and its recognition of the promoter from -27 to -16. Core Factor's intrinsic mobility correlates well with different conformational states of the Pol I cleft, in addition to the stabilization of either Rrn7 N-terminal domain near Pol I wall or the tandem winged helix domain of A49 at a partially overlapping location. Comparison of the three states in this study with the Pol II system suggests that a ratchet motion of the Core Factor-DNA sub-complex at upstream facilitates promoter melting in an ATP-independent manner, distinct from a DNA translocase actively threading the downstream DNA in the Pol II PIC.
History
DepositionJun 16, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 2, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Nov 8, 2017Group: Derived calculations / Category: pdbx_struct_assembly
Item: _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.4Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase I subunit RPA190
B: DNA-directed RNA polymerase I subunit RPA135
C: DNA-directed RNA polymerases I and III subunit RPAC1
D: DNA-directed RNA polymerase I subunit RPA14
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 I subunit RPA43
H: DNA-directed RNA polymerases I, II, and III subunit RPABC3
I: DNA-directed RNA polymerase I subunit RPA12
J: DNA-directed RNA polymerases I, II, and III subunit RPABC5
K: DNA-directed RNA polymerases I and III subunit RPAC2
L: DNA-directed RNA polymerases I, II, and III subunit RPABC4
M: DNA-directed RNA polymerase I subunit RPA49
N: DNA-directed RNA polymerase I subunit RPA34
O: RNA polymerase I-specific transcription initiation factor RRN6
P: RNA polymerase I-specific transcription initiation factor RRN7
Q: RNA polymerase I-specific transcription initiation factor RRN11
R: RNA
S: non-template strand DNA
T: template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)846,27627
Polymers845,81820
Non-polymers4587
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area115320 Å2
ΔGint-663 kcal/mol
Surface area241990 Å2

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Components

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DNA-directed RNA polymerase I subunit ... , 7 types, 7 molecules ABDGIMN

#1: Protein DNA-directed RNA polymerase I subunit RPA190 / DNA-directed RNA polymerase I 190 kDa polypeptide / A190 / DNA-directed RNA polymerase I largest subunit


Mass: 186676.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P10964, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase I subunit RPA135 / DNA-directed RNA polymerase I 135 kDa polypeptide / A135 / DNA-directed RNA polymerase I ...DNA-directed RNA polymerase I 135 kDa polypeptide / A135 / DNA-directed RNA polymerase I polypeptide 2 / RNA polymerase I subunit 2


Mass: 135910.328 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P22138, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase I subunit RPA14 / A14 / DNA-directed RNA polymerase I 14 kDa polypeptide


Mass: 14599.128 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P50106
#7: Protein DNA-directed RNA polymerase I subunit RPA43 / A43 / DNA-directed DNA-dependent RNA polymerase 36 kDa polypeptide


Mass: 36264.852 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P46669
#9: Protein DNA-directed RNA polymerase I subunit RPA12 / A12 / A12.2 / DNA-directed RNA polymerase I 13.7 kDa polypeptide


Mass: 13676.566 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P32529
#13: Protein DNA-directed RNA polymerase I subunit RPA49 / A49 / DNA-directed RNA polymerase I 49 kDa polypeptide


Mass: 46721.707 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q01080
#14: Protein DNA-directed RNA polymerase I subunit RPA34 / A34 / DNA-directed DNA-dependent RNA polymerase 34.5 kDa polypeptide / A34.5


Mass: 26933.518 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P47006

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DNA-directed RNA polymerases I and III subunit ... , 2 types, 2 molecules CK

#3: Protein DNA-directed RNA polymerases I and III subunit RPAC1 / RNA polymerases I and III subunit AC1 / C37 / DNA-directed RNA polymerases I and III 40 kDa polypeptide / C40


Mass: 37732.613 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P07703
#11: Protein DNA-directed RNA polymerases I and III subunit RPAC2 / RNA polymerases I and III subunit AC2 / AC19 / DNA-directed RNA polymerases I and III 16 kDa ...RNA polymerases I and III subunit AC2 / AC19 / DNA-directed RNA polymerases I and III 16 kDa polypeptide / RPA19


Mass: 16167.860 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P28000

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DNA-directed RNA polymerases I, II, and III subunit ... , 5 types, 5 molecules EFHJL

#5: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC1 / RNA polymerases I / II / and III subunit ABC1 / ABC27 / DNA-directed RNA polymerases I / and III 27 ...RNA polymerases I / II / and III subunit ABC1 / ABC27 / DNA-directed RNA polymerases I / and III 27 kDa polypeptide


Mass: 25117.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P20434
#6: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC2 / RNA polymerases I / II / and III subunit ABC2 / ABC23 / DNA-directed RNA polymerases I / and III 23 ...RNA polymerases I / II / and III subunit ABC2 / ABC23 / DNA-directed RNA polymerases I / and III 23 kDa polypeptide


Mass: 17931.834 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P20435
#8: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC3 / 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 / and III 14.5 kDa polypeptide


Mass: 16525.363 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P20436
#10: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC5 / RNA polymerases I / II / and III subunit ABC5 / ABC10-beta / ABC8 / DNA-directed RNA polymerases I ...RNA polymerases I / II / and III subunit ABC5 / ABC10-beta / ABC8 / DNA-directed RNA polymerases I / and III 8.3 kDa polypeptide


Mass: 8290.732 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P22139
#12: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerases I / II / and III subunit ABC4 / ABC10-alpha


Mass: 7729.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P40422

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RNA polymerase I-specific transcription initiation factor ... , 3 types, 3 molecules OPQ

#15: Protein RNA polymerase I-specific transcription initiation factor RRN6


Mass: 100690.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RRN6, YBL014C, YBL0311, YBL0312 / Production host: Escherichia coli (E. coli) / References: UniProt: P32786
#16: Protein RNA polymerase I-specific transcription initiation factor RRN7


Mass: 60435.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RRN7, YJL025W, J1273 / Production host: Escherichia coli (E. coli) / References: UniProt: P40992
#17: Protein RNA polymerase I-specific transcription initiation factor RRN11


Mass: 59334.262 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RRN11, YML043C, YM9827.09C / Production host: Escherichia coli (E. coli) / References: UniProt: Q04712

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DNA chain , 2 types, 2 molecules ST

#19: DNA chain non-template strand DNA


Mass: 16803.787 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288c (yeast)
#20: DNA chain template strand DNA


Mass: 16360.488 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288c (yeast)

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RNA chain / Non-polymers , 2 types, 8 molecules R

#18: RNA chain RNA


Mass: 1915.213 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288c (yeast)
#21: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn

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Details

Sequence detailsDUE TO LOW RESOLUTION THE SEQUENCE CORRESPONDING TO UNK SEGMENT COULD NOT BE MODELED

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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: RNA Polymerase I Initial Transcribing Complex State 2 / Type: COMPLEX / Entity ID: #1-#20 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.9 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil S7/2
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 22500 X / Calibrated defocus min: 1500 nm / Calibrated defocus max: 4500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 56.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon3.1image acquisition
4Gctf0.5CTF correction
7NAMD2.8model fittingMDFF flexible fitting
8UCSF Chimera1.11.2model fittingrigid body fitting
10Coot0.8.8model refinement
11PHENIX1.10.1model refinement
12RELION2initial Euler assignment
13RELION2final Euler assignment
14RELION2classification
15RELION23D reconstruction
CTF correctionDetails: CTF amplitude correction was performed following 3D auto refinement in relion.
Type: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38340 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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