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- EMDB-28149: Mycobacterium tuberculosis paused transcription complex with Baci... -

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Basic information

Entry
Database: EMDB / ID: EMD-28149
TitleMycobacterium tuberculosis paused transcription complex with Bacillus subtilis NusG
Map data
Sample
  • Complex: Paused transcription complex with NusG
    • 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: Transcription termination/antitermination protein NusG
    • DNA: DNA (38-MER)
    • DNA: DNA (38-MER)
    • RNA: RNA (29-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsTranscription elongation RNA polymerase pausing NusG cryo-EM / TRANSCRIPTION
Function / homology
Function and homology information


Antimicrobial action and antimicrobial resistance in Mtb / transcription elongation-coupled chromatin remodeling / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...Antimicrobial action and antimicrobial resistance in Mtb / transcription elongation-coupled chromatin remodeling / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / response to antibiotic / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
: / Transcription antitermination protein, NusG / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription termination factor nusG signature. / NusG-like / Transcription termination factor nusG / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / NusG, N-terminal domain superfamily / DNA-directed RNA polymerase, omega subunit ...: / Transcription antitermination protein, NusG / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription termination factor nusG signature. / NusG-like / Transcription termination factor nusG / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / NusG, N-terminal domain superfamily / 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 / 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 / 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 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 / KOW (Kyprides, Ouzounis, Woese) motif. / Translation protein SH3-like domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha / Transcription termination/antitermination protein NusG
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria) / Bacillus subtilis subsp. subtilis str. 168 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsVishwakarma RK / Murakami KS
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131860 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM098399 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.
Authors: Rishi K Vishwakarma / M Zuhaib Qayyum / Paul Babitzke / Katsuhiko S Murakami /
Abstract: NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including by sequence-specific interaction with a conserved pause-inducing TTNTTT motif found in the ...NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including by sequence-specific interaction with a conserved pause-inducing TTNTTT motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the β-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.
History
DepositionSep 14, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_28149.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 400 pix.
= 348. Å
0.87 Å/pix.
x 400 pix.
= 348. Å
0.87 Å/pix.
x 400 pix.
= 348. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 0.238
Minimum - Maximum-0.7183045 - 2.1008275
Average (Standard dev.)0.0043592723 (±0.064726435)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 348.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_28149_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_28149_half_map_2.map
Projections & Slices
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Sample components

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Entire : Paused transcription complex with NusG

EntireName: Paused transcription complex with NusG
Components
  • Complex: Paused transcription complex with NusG
    • 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: Transcription termination/antitermination protein NusG
    • DNA: DNA (38-MER)
    • DNA: DNA (38-MER)
    • RNA: RNA (29-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Paused transcription complex with NusG

SupramoleculeName: Paused transcription complex with NusG / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: It contains M. tuberculosis RNA polymerase subunits and B. subtilis NusG
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)

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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: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 37.745328 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLISQRPTLS EDVLTDNRSQ FVIEPLEPGF GYTLGNSLRR TLLSSIPGAA VTSIRIDGVL HEFTTVPGVK EDVTEIILNL KSLVVSSEE DEPVTMYLRK QGPGEVTAGD IVPPAGVTVH NPGMHIATLN DKGKLEVELV VERGRGYVPA VQNRASGAEI G RIPVDSIY ...String:
MLISQRPTLS EDVLTDNRSQ FVIEPLEPGF GYTLGNSLRR TLLSSIPGAA VTSIRIDGVL HEFTTVPGVK EDVTEIILNL KSLVVSSEE DEPVTMYLRK QGPGEVTAGD IVPPAGVTVH NPGMHIATLN DKGKLEVELV VERGRGYVPA VQNRASGAEI G RIPVDSIY SPVLKVTYKV DATRVEQRTD FDKLILDVET KNSISPRDAL ASAGKTLVEL FGLARELNVE AEGIEIGPSP AE ADHIASF ALPIDDLDLT VRSYNCLKRE GVHTVGELVA RTESDLLDIR NFGQKSIDEV KIKLHQLGLS LKDSPPSFDP SEV AGYDVA TGTWSTEGAY DEQDYAETEQ L

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: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 130.018828 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLEGCILADS RQSKTAASPS PSRPQSSSNN SVPGAPNRVS FAKLREPLEV PGLLDVQTDS FEWLIGSPRW RESAAERGDV NPVGGLEEV LYELSPIEDF SGSMSLSFSD PRFDDVKAPV DECKDKDMTY AAPLFVTAEF INNNTGEIKS QTVFMGDFPM M TEKGTFII ...String:
MLEGCILADS RQSKTAASPS PSRPQSSSNN SVPGAPNRVS FAKLREPLEV PGLLDVQTDS FEWLIGSPRW RESAAERGDV NPVGGLEEV LYELSPIEDF SGSMSLSFSD PRFDDVKAPV DECKDKDMTY AAPLFVTAEF INNNTGEIKS QTVFMGDFPM M TEKGTFII NGTERVVVSQ LVRSPGVYFD ETIDKSTDKT LHSVKVIPSR GAWLEFDVDK RDTVGVRIDR KRRQPVTVLL KA LGWTSEQ IVERFGFSEI MRSTLEKDNT VGTDEALLDI YRKLRPGEPP TKESAQTLLE NLFFKEKRYD LARVGRYKVN KKL GLHVGE PITSSTLTEE DVVATIEYLV RLHEGQTTMT VPGGVEVPVE TDDIDHFGNR RLRTVGELIQ NQIRVGMSRM ERVV RERMT TQDVEAITPQ TLINIRPVVA AIKEFFGTSQ LSQFMDQNNP LSGLTHKRRL SALGPGGLSR ERAGLEVRDV HPSHY GRMC PIETPEGPNI GLIGSLSVYA RVNPFGFIET PYRKVVDGVV SDEIVYLTAD EEDRHVVAQA NSPIDADGRF VEPRVL VRR KAGEVEYVPS SEVDYMDVSP RQMVSVATAM IPFLEHDDAN RALMGANMQR QAVPLVRSEA PLVGTGMELR AAIDAGD VV VAEESGVIEE VSADYITVMH DNGTRRTYRM RKFARSNHGT CANQCPIVDA GDRVEAGQVI ADGPCTDDGE MALGKNLL V AIMPWEGHNY EDAIILSNRL VEEDVLTSIH IEEHEIDARD TKLGAEEITR DIPNISDEVL ADLDERGIVR IGAEVRDGD ILVGKVTPKG ETELTPEERL LRAIFGEKAR EVRDTSLKVP HGESGKVIGI RVFSREDEDE LPAGVNELVR VYVAQKRKIS DGDKLAGRH GNKGVIGKIL PVEDMPFLAD GTPVDIILNT HGVPRRMNIG QILETHLGWC AHSGWKVDAA KGVPDWAARL P DELLEAQP NAIVSTPVFD GAQEAELQGL LSCTLPNRDG DVLVDADGKA MLFDGRSGEP FPYPVTVGYM YIMKLHHLVD DK IHARSTG PYSMITQQPL GGKAQFGGQR FGEMECWAMQ AYGAAYTLQE LLTIKSDDTV GRVKVYEAIV KGENIPEPGI PES FKVLLK ELQSLCLNVE VLSSDGAAIE LREGEDEDLE RAAANLGINL SRNESASVED LA

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: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 146.968969 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLDVNFFDEL RIGLATAEDI RQWSYGEVKK PETINYRTLK PEKDGLFCEK IFGPTRDWEC YCGKYKRVRF KGIICERCGV EVTRAKVRR ERMGHIELAA PVTHIWYFKG VPSRLGYLLD LAPKDLEKII YFAAYVITSV DEEMRHNELS TLEAEMAVER K AVEDQRDG ...String:
MLDVNFFDEL RIGLATAEDI RQWSYGEVKK PETINYRTLK PEKDGLFCEK IFGPTRDWEC YCGKYKRVRF KGIICERCGV EVTRAKVRR ERMGHIELAA PVTHIWYFKG VPSRLGYLLD LAPKDLEKII YFAAYVITSV DEEMRHNELS TLEAEMAVER K AVEDQRDG ELEARAQKLE ADLAELEAEG AKADARRKVR DGGEREMRQI RDRAQRELDR LEDIWSTFTK LAPKQLIVDE NL YRELVDR YGEYFTGAMG AESIQKLIEN FDIDAEAESL RDVIRNGKGQ KKLRALKRLK VVAAFQQSGN SPMGMVLDAV PVI PPELRP MVQLDGGRFA TSDLNDLYRR VINRNNRLKR LIDLGAPEII VNNEKRMLQE SVDALFDNGR RGRPVTGPGN RPLK SLSDL LKGKQGRFRQ NLLGKRVDYS GRSVIVVGPQ LKLHQCGLPK LMALELFKPF VMKRLVDLNH AQNIKSAKRM VERQR PQVW DVLEEVIAEH PVLLNRAPTL HRLGIQAFEP MLVEGKAIQL HPLVCEAFNA DFDGDQMAVH LPLSAEAQAE ARILML SSN NILSPASGRP LAMPRLDMVT GLYYLTTEVP GDTGEYQPAS GDHPETGVYS SPAEAIMAAD RGVLSVRAKI KVRLTQL RP PVEIEAELFG HSGWQPGDAW MAETTLGRVM FNELLPLGYP FVNKQMHKKV QAAIINDLAE RYPMIVVAQT VDKLKDAG F YWATRSGVTV SMADVLVPPR KKEILDHYEE RADKVEKQFQ RGALNHDERN EALVEIWKEA TDEVGQALRE HYPDDNPII TIVDSGATGN FTQTRTLAGM KGLVTNPKGE FIPRPVKSSF REGLTVLEYF INTHGARKGL ADTALRTADS GYLTRRLVDV SQDVIVREH DCQTERGIVV ELAERAPDGT LIRDPYIETS AYARTLGTDA VDEAGNVIVE RGQDLGDPEI DALLAAGITQ V KVRSVLTC ATSTGVCATC YGRSMATGKL VDIGEAVGIV AAQSIGEPGT QLTMRTFHQG GVGEDITGGL PRVQELFEAR VP RGKAPIA DVTGRVRLED GERFYKITIV PDDGGEEVVY DKISKRQRLR VFKHEDGSER VLSDGDHVEV GQQLMEGSAD PHE VLRVQG PREVQIHLVR EVQEVYRAQG VSIHDKHIEV IVRQMLRRVT IIDSGSTEFL PGSLIDRAEF EAENRRVVAE GGEP AAGRP VLMGITKASL ATDSWLSAAS FQETTRVLTD AAINCRSDKL NGLKENVIIG KLIPAGTGIN RYRNIAVQPT EEARA AAYT IPSYEDQYYS PDFGAATGAA VPLDDYGYSD YR

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: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 11.85114 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSISQSDASL AAVPAVDQFD PSSGASGGYD TPLGITNPPI DELLDRVSSK YALVIYAAKR ARQINDYYNQ LGEGILEYVG PLVEPGLQE KPLSIALREI HADLLEHTEG E

UniProtKB: DNA-directed RNA polymerase subunit omega

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Macromolecule #5: Transcription termination/antitermination protein NusG

MacromoleculeName: Transcription termination/antitermination protein NusG
type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Strain: 168
Molecular weightTheoretical: 20.154135 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MEKNWYVVHT YSGYENKVKA NLEKRVESMG MQDKIFRVVV PEEEETDIKN GKKKVVKKKV FPGYVLVEIV MTDDSWYVVR NTPGVTGFV GSAGSGSKPT PLLPGEAETI LKRMGMDERK TDIDFELKET VKVIDGPFAN FTGSIEEIDY DKSKVKVFVN M FGRETPVE LEFTQIDKL

UniProtKB: Transcription termination/antitermination protein NusG

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Macromolecule #6: DNA (38-MER)

MacromoleculeName: DNA (38-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.180794 KDa
SequenceString:
(DC)(DG)(DG)(DC)(DA)(DG)(DT)(DC)(DG)(DC) (DC)(DG)(DT)(DC)(DT)(DA)(DC)(DC)(DT)(DC) (DT)(DC)(DC)(DA)(DA)(DG)(DA)(DG)(DC) (DA)(DG)(DC)(DA)(DT)(DG)(DC)(DG)(DC)(DC) (DC)

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Macromolecule #7: DNA (38-MER)

MacromoleculeName: DNA (38-MER) / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.248812 KDa
SequenceString:
(DG)(DG)(DG)(DC)(DG)(DC)(DA)(DT)(DG)(DC) (DT)(DG)(DC)(DT)(DC)(DT)(DT)(DC)(DT)(DT) (DT)(DG)(DC)(DC)(DA)(DT)(DC)(DA)(DC) (DG)(DG)(DC)(DG)(DA)(DC)(DT)(DG)(DC)(DC) (DG)

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Macromolecule #8: RNA (29-MER)

MacromoleculeName: RNA (29-MER) / type: rna / ID: 8 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.700808 KDa
SequenceString:
UCCGAAGCUU CGGCUUCGGC AGGAGAGGUA

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / 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

BufferpH: 7.9
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: Blot for 4-5 seconds before plunging in liquid ethane.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 110849
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING

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