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- EMDB-24424: Cryo-EM structure of Thermus thermophilus reiterative transcripti... -

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Basic information

Entry
Database: EMDB / ID: EMD-24424
TitleCryo-EM structure of Thermus thermophilus reiterative transcription complex with 11nt oligo-G RNA
Map data
Sample
  • Complex: reiterative transcription initiation complex with 11nt oligo-G RNA
    • Protein or peptide: DNA-directed RNA polymerase subunit alpha
    • Protein or peptide: DNA-directed RNA polymerase subunit beta
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'
    • Protein or peptide: DNA-directed RNA polymerase subunit omega
    • Protein or peptide: RNA polymerase sigma factor SigA
    • DNA: DNA (31-MER) nontemplate strand
    • DNA: DNA (31-MER) template strand
    • RNA: RNA (5'-R(P*GP*GP*GP*GP*GP*GP*GP*GP*GP*GP*G)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsThermus thermophilus / transcription initiation / TRANSCRIPTION
Function / homology
Function and homology information


sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding ...sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / cytoplasm
Similarity search - Function
: / DNA-directed RNA polymerase subunit beta', hybrid domain / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. ...: / DNA-directed RNA polymerase subunit beta', hybrid domain / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / 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 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsLiu Y / Ebright RH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Structural and mechanistic basis of reiterative transcription initiation.
Authors: Yu Liu / Libing Yu / Chirangini Pukhrambam / Jared T Winkelman / Emre Firlar / Jason T Kaelber / Yu Zhang / Bryce E Nickels / Richard H Ebright /
Abstract: Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5' sequences noncomplementary to the ...Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5' sequences noncomplementary to the DNA template. Here, using crystallography and cryoelectron microscopy to define structures, protein-DNA photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and single-molecule DNA nanomanipulation to assess RNA polymerase (RNAP)-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation 1) occurs without DNA scrunching; 2) involves a short, 2- to 3-bp, RNA-DNA hybrid; and 3) generates RNA that exits RNAP through the portal by which scrunched nontemplate-strand DNA exits RNAP in standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with slipping of RNA relative to DNA within a short RNA-DNA hybrid, and with extrusion of RNA from RNAP through an alternative RNA exit.
History
DepositionJul 10, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-7rdq
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24424.png

Downloads & links

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Map

FileDownload / File: emd_24424.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 265.728 Å		1.04 Å/pix.
x 256 pix.
= 265.728 Å		1.04 Å/pix.
x 256 pix.
= 265.728 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.038 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.09103123 - 0.19569632
Average (Standard dev.)-0.000109607565 (±0.0063472446)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 265.728 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0381.0381.038
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z265.728265.728265.728
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0910.196-0.000

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Supplemental data

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Sample components

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Entire : reiterative transcription initiation complex with 11nt oligo-G RNA

EntireName: reiterative transcription initiation complex with 11nt oligo-G RNA
Components
  • Complex: reiterative transcription initiation complex with 11nt oligo-G RNA
    • Protein or peptide: DNA-directed RNA polymerase subunit alpha
    • Protein or peptide: DNA-directed RNA polymerase subunit beta
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'
    • Protein or peptide: DNA-directed RNA polymerase subunit omega
    • Protein or peptide: RNA polymerase sigma factor SigA
    • DNA: DNA (31-MER) nontemplate strand
    • DNA: DNA (31-MER) template strand
    • RNA: RNA (5'-R(P*GP*GP*GP*GP*GP*GP*GP*GP*GP*GP*G)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: reiterative transcription initiation complex with 11nt oligo-G RNA

SupramoleculeName: reiterative transcription initiation complex with 11nt oligo-G RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 425 KDa

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Macromolecule #1: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 35.056164 KDa
SequenceString: MLDSKLKAPV FTVRTQGREY GEFVLEPLER GFGVTLGNPL RRILLSSIPG TAVTSVYIED VLHEFSTIPG VKEDVVEIIL NLKELVVRF LNPSLQTVTL LLKAEGPKEV KARDFLPVAD VEIMNPDLHI ATLEEGGRLN MEVRVDRGVG YVPAEKHGIK D RINAIPVD ...String:
MLDSKLKAPV FTVRTQGREY GEFVLEPLER GFGVTLGNPL RRILLSSIPG TAVTSVYIED VLHEFSTIPG VKEDVVEIIL NLKELVVRF LNPSLQTVTL LLKAEGPKEV KARDFLPVAD VEIMNPDLHI ATLEEGGRLN MEVRVDRGVG YVPAEKHGIK D RINAIPVD AVFSPVRRVA FQVEDTRLGQ RTDLDKLTLR IWTDGSVTPL EALNQAVEIL REHLTYFSNP QAAAVAAPEE AK EPEAPPE QEEELDLPLE ELGLSTRVLH SLKEEGIESV RALLALNLKD LKNIPGIGER SLEEIKEALE KKGFTLKE

UniProtKB: DNA-directed RNA polymerase subunit alpha

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Macromolecule #2: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 125.436539 KDa
SequenceString: MEIKRFGRIR EVIPLPPLTE IQVESYRRAL QADVPPEKRE NVGIQAAFRE TFPIEEEDKG KGGLVLDFLE YRLGEPPFPQ DECREKDLT YQAPLYARLQ LIHKDTGLIK EDEVFLGHIP LMTEDGSFII NGADRVIVSQ IHRSPGVYFT PDPARPGRYI A SIIPLPKR ...String:
MEIKRFGRIR EVIPLPPLTE IQVESYRRAL QADVPPEKRE NVGIQAAFRE TFPIEEEDKG KGGLVLDFLE YRLGEPPFPQ DECREKDLT YQAPLYARLQ LIHKDTGLIK EDEVFLGHIP LMTEDGSFII NGADRVIVSQ IHRSPGVYFT PDPARPGRYI A SIIPLPKR GPWIDLEVEP NGVVSMKVNK RKFPLVLLLR VLGYDQETLA RELGAYGELV QGLMDESVFA MRPEEALIRL FT LLRPGDP PKRDKAVAYV YGLIADPRRY DLGEAGRYKA EEKLGIRLSG RTLARFEDGE FKDEVFLPTL RYLFALTAGV PGH EVDDID HLGNRRIRTV GELMTDQFRV GLARLARGVR ERMLMGSEDS LTPAKLVNSR PLEAAIREFF SRSQLSQFKD ETNP LSSLR HKRRISALGP GGLTRERAGF DVRDVHRTHY GRICPVETPE GANIGLITSL AAYARVDELG FIRTPYRRVV GGVVT DEVV YMTATEEDRY TIAQANTPLE GNRIAAERVV ARRKGEPVIV SPEEVEFMDV SPKQVFSVNT NLIPFLEHDD ANRALM GSN MQTQAVPLIR AQAPVVMTGL EERVVRDSLA ALYAEEDGEV AKVDGNRIVV RYEDGRLVEY PLRRFYRSNQ GTALDQR PR VVVGQRVRKG DLLADGPASE NGFLALGQNV LVAIMPFDGY NFEDAIVISE ELLKRDFYTS IHIERYEIEA RDTKLGPE R ITRDIPHLSE AALRDLDEEG VVRIGAEVKP GDILVGRTSF KGESEPTPEE RLLRSIFGEK ARDVKDTSLR VPPGEGGIV VRTVRLRRGD PGVELKPGVR EVVRVYVAQK RKLQVGDKLA NRHGNKGVVA KILPVEDMPH LPDGTPVDVI LNPLGVPSRM NLGQILETH LGLAGYFLGQ RYISPIFDGA KEPEIKELLA QAFEVYFGKR KGEGFGVDKR EVEVLRRAEK LGLVTPGKTP E EQLKELFL QGKVVLYDGR TGEPIEGPIV VGQMFIMKLY HMVEDKMHAR STGPYSLITQ QPLGGKAQFG GQRFGEMEVW AL EAYGAAH TLQEMLTLKS DDIEGRNAAY EAIIKGEDVP EPSVPESFRV LVKELQALAL DVQTLDEKDN PVDIFEGLAS KR

UniProtKB: DNA-directed RNA polymerase subunit beta

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Macromolecule #3: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 170.997391 KDa
SequenceString: MKKEVRKVRI ALASPEKIRS WSYGEVEKPE TINYRTLKPE RDGLFDERIF GPIKDYECAC GKYKRQRFEG KVCERCGVEV TKSIVRRYR MGHIELATPA AHIWFVKDVP SKIGTLLDLS ATELEQVLYF SKYIVLDPKG AILNGVPVEK RQLLTDEEYR E LRYGKQET ...String:
MKKEVRKVRI ALASPEKIRS WSYGEVEKPE TINYRTLKPE RDGLFDERIF GPIKDYECAC GKYKRQRFEG KVCERCGVEV TKSIVRRYR MGHIELATPA AHIWFVKDVP SKIGTLLDLS ATELEQVLYF SKYIVLDPKG AILNGVPVEK RQLLTDEEYR E LRYGKQET YPLPPGVDAL VKDGEEVVKG QELAPGVVSR LDGVALYRFP RRVRVEYVKK ERAGLRLPLA AWVEKEAYKP GE ILAELPE PYLFRAEEEG VVELKELEEG AFLVLRREDE PVATYFLPVG MTPLVVHGEI VEKGQPLAEA KGLLRMPRQV RAA QVEAEE EGETVYLTLF LEWTEPKDYR VQPHMNVVVP EGARVEAGDK IVAAIDPEEE VIAEAEGVVH LHEPASILVV KARV YPFED DVEVSTGDRV APGDVLADGG KVKSDVYGRV EVDLVRNVVR VVESYDIDAR MGAEAIQQLL KELDLEALEK ELLEE MKHP SRARRAKARK RLEVVRAFLD SGNRPEWMIL EAVPVLPPDL RPMVQVDGGR FATSDLNDLY RRLINRNNRL KKLLAQ GAP EIIIRNEKRM LQEAVDALLD NGRRGAPVTN PGSDRPLRSL TDILSGKQGR FRQNLLGKRV DYSGRSVIVV GPQLKLH QC GLPKRMALEL FKPFLLKKME EKGIAPNVKA ARRMLERQRD IKDEVWDALE EVIHGKVVLL NRAPTLHRLG IQAFQPVL V EGQSIQLHPL VCEAFNADFD GDQMAVHVPL SSFAQAEARI QMLSAHNLLS PASGEPLAKP SRDIILGLYY ITQVRKEKK GAGLEFATPE EALAAHERGE VALNAPIKVA GRETSVGRLK YVFANPDEAL LAVAHGIVDL QDVVTVRYMG KRLETSPGRI LFARIVAEA VEDEKVAWEL IQLDVPQEKN SLKDLVYQAF LRLGMEKTAR LLDALKYYGF TFSTTSGITI GIDDAVIPEE K KQYLEEAD RKLLQIEQAY EMGFLTDRER YDQILQLWTE TTEKVTQAVF KNFEENYPFN PLYVMAQSGA RGNPQQIRQL CG LRGLMQK PSGETFEVPV RSSFREGLTV LEYFISSHGA RKGGADTALR TADSGYLTRK LVDVTHEIVV READCGTTNY ISV PLFQPD EVTRSLRLRK RADIEAGLYG RVLAREVEVL GVRLEEGRYL SMDDVHLLIK AAEAGEIQEV PVRSPLTCQT RYGV CQKCY GYDLSMARPV SIGEAVGIVA AQSIGEPGTQ LTMRTFHTGG VAGAADITQG LPRVIELFEA RRPKAKAVIS EIDGV VRIE ETEEKLSVFV ESEGFSKEYK LPKEARLLVK DGDYVEAGQP LTRGAIDPHQ LLEAKGPEAV ERYLVEEIQK VYRAQG VKL HDKHIEIVVR QMMKYVEVTD PGDSRLLEGQ VLEKWDVEAL NERLIAEGKT PVAWKPLLMG VTKSALSTKS WLSAASF QN TTHVLTEAAI AGKKDELIGL KENVILGRLI PAGTGSDFVR FTQVVDQKTL KAIEEARKEA VEAKERPAAR RGVKREQP G KQA

UniProtKB: DNA-directed RNA polymerase subunit beta'

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Macromolecule #4: DNA-directed RNA polymerase subunit omega

MacromoleculeName: DNA-directed RNA polymerase subunit omega / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 11.533316 KDa
SequenceString:
MAEPGIDKLF GMVDSKYRLT VVVAKRAQQL LRHGFKNTVL EPEERPKMQT LEGLFDDPNA VTWAMKELLT GRLVFGENLV PEDRLQKEM ERLYPVEREE

UniProtKB: DNA-directed RNA polymerase subunit omega

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Macromolecule #5: RNA polymerase sigma factor SigA

MacromoleculeName: RNA polymerase sigma factor SigA / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 48.598031 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKSKRKNAQ AQEAQETEVL VQEEAEELPE FPEGEPDPDL EDPDLTLEDD LLDLPEEGEG LDLEEEEEDL PIPKISTSDP VRQYLHEIG QVPLLTLEEE VELARKVEEG MEAIKKLSEI TGLDPDLIRE VVRAKILGSA RVRHIPGLKE TLDPKTVEEI D QKLKSLPK ...String:
MKKSKRKNAQ AQEAQETEVL VQEEAEELPE FPEGEPDPDL EDPDLTLEDD LLDLPEEGEG LDLEEEEEDL PIPKISTSDP VRQYLHEIG QVPLLTLEEE VELARKVEEG MEAIKKLSEI TGLDPDLIRE VVRAKILGSA RVRHIPGLKE TLDPKTVEEI D QKLKSLPK EHKRYLHIAR EGEAARQHLI EANLRLVVSI AKKYTGRGLS FLDLIQEGNQ GLIRAVEKFE YKRRFKFSTY AT WWIRQAI NRAIADQART IRIPVHMVET INKLSRTARQ LQQELGREPT YEEIAEAMGP GWDAKRVEET LKIAQEPVSL ETP IGDEKD SFYGDFIPDE HLPSPVDAAT QSLLSEELEK ALSKLSEREA MVLKLRKGLI DGREHTLEEV GAFFGVTRER IRQI ENKAL RKLKYHESRT RKLRDFLD

UniProtKB: RNA polymerase sigma factor SigA

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Macromolecule #6: DNA (31-MER) nontemplate strand

MacromoleculeName: DNA (31-MER) nontemplate strand / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.331629 KDa
SequenceString:
(DG)(DT)(DG)(DT)(DG)(DC)(DT)(DA)(DT)(DA) (DA)(DT)(DG)(DG)(DG)(DA)(DG)(DC)(DT)(DG) (DG)(DC)(DA)(DC)(DG)(DG)(DA)(DT)(DG) (DC)(DA)(DG)(DG)

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Macromolecule #7: DNA (31-MER) template strand

MacromoleculeName: DNA (31-MER) template strand / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.1005 KDa
SequenceString:
(DC)(DC)(DT)(DG)(DC)(DA)(DT)(DC)(DC)(DG) (DT)(DG)(DC)(DC)(DC)(DT)(DG)(DA)(DG)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DC) (DA)(DC)(DA)(DC)

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Macromolecule #8: RNA (5'-R(P*GP*GP*GP*GP*GP*GP*GP*GP*GP*GP*G)-3')

MacromoleculeName: RNA (5'-R(P*GP*GP*GP*GP*GP*GP*GP*GP*GP*GP*G)-3') / type: rna / ID: 8 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.752306 KDa
SequenceString:
GGGGGGGGGG G

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.69 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris hydrochloride
100.0 mMNaClsodium chloride
5.0 mMMgCl2Magnesium chloride
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.027 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Number real images: 3424 / Average exposure time: 0.2 sec. / Average electron dose: 0.994 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -2.8000000000000003 µm / Nominal defocus min: -1.2 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2143284
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4g7h

Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 339426
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final 3D classificationNumber classes: 5 / Avg.num./class: 100000 / Software - Name: RELION (ver. 3.1.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4g7h


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7rdq:
Cryo-EM structure of Thermus thermophilus reiterative transcription complex with 11nt oligo-G RNA

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Atomic model buiding 2

Initial modelPDB ID:

4g7h


Chain - Chain ID: B / Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7rdq:
Cryo-EM structure of Thermus thermophilus reiterative transcription complex with 11nt oligo-G RNA

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Atomic model buiding 3

Initial modelPDB ID:

4g7h


Chain - Chain ID: C / Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7rdq:
Cryo-EM structure of Thermus thermophilus reiterative transcription complex with 11nt oligo-G RNA

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Atomic model buiding 4

Initial modelPDB ID:

4g7h


Chain - Chain ID: D / Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7rdq:
Cryo-EM structure of Thermus thermophilus reiterative transcription complex with 11nt oligo-G RNA

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Atomic model buiding 5

Initial modelPDB ID:

4g7h


Chain - Chain ID: E / Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7rdq:
Cryo-EM structure of Thermus thermophilus reiterative transcription complex with 11nt oligo-G RNA

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Atomic model buiding 6

Initial modelPDB ID:

4g7h


Chain - Chain ID: F / Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7rdq:
Cryo-EM structure of Thermus thermophilus reiterative transcription complex with 11nt oligo-G RNA

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