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- EMDB-16929: Cryo-EM structure of Pyrococcus furiosus transcription elongation... -

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Basic information

Entry
Database: EMDB / ID: EMD-16929
TitleCryo-EM structure of Pyrococcus furiosus transcription elongation complex bound to Spt4/5
Map data
Sample
  • Complex: Transcription elongation complex with Spt4/5
    • Complex: Pyrococcus furiosus RNA polymerase
      • Protein or peptide: x 11 types
    • Complex: Spt4/5
      • Protein or peptide: x 2 types
    • Complex: Nucleotide strands
      • DNA: x 2 types
      • RNA: x 1 types
  • Ligand: x 2 types
KeywordsRNA / Polymerase / Elongation complex / Pyrococcus furiosus / DNA / Archaea / TRANSCRIPTION
Function / homology
Function and homology information


transcription elongation-coupled chromatin remodeling / translation elongation factor activity / transcription elongation by RNA polymerase I / tRNA transcription by RNA polymerase III / DNA-directed RNA polymerase activity / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / ribonucleoside binding / : ...transcription elongation-coupled chromatin remodeling / translation elongation factor activity / transcription elongation by RNA polymerase I / tRNA transcription by RNA polymerase III / DNA-directed RNA polymerase activity / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / protein dimerization activity / ribosome / structural constituent of ribosome / nucleotide binding / mRNA binding / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
Archaeal transcription elongation factor Spt4 / Transcription elongation factor Spt5, archaeal / Archaeal transcription elongation factor Spt4 superfamily / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo7 / HRDC domain superfamily ...Archaeal transcription elongation factor Spt4 / Transcription elongation factor Spt5, archaeal / Archaeal transcription elongation factor Spt4 superfamily / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo7 / HRDC domain superfamily / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / DNA-directed RNA polymerase, subunit E/RPC8 / NGN domain / Transcription elongation factor SPT5 / Early transcription elongation factor of RNA pol II, NGN section / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / NusG, N-terminal domain superfamily / S1 domain profile. / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / 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 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / : / 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, 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. / 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 / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / 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. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / 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 Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, RBP11-like subunit / 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.
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo12 / Transcription elongation factor Spt5 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo4 ...DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo12 / Transcription elongation factor Spt5 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo7 / Transcription elongation factor Spt4 / DNA-directed RNA polymerase subunit Rpo11
Similarity search - Component
Biological speciesPyrococcus furiosus DSM 3638 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsTarau DM / Reichelt R / Heiss FB / Pilsl M / Hausner W / Engel C / Grohmann D
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB960 Germany
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.
Authors: Daniela Tarău / Felix Grünberger / Michael Pilsl / Robert Reichelt / Florian Heiß / Sabine König / Henning Urlaub / Winfried Hausner / Christoph Engel / Dina Grohmann /
Abstract: Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, ...Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.
History
DepositionMar 28, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16929.png

Downloads & links

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Map

FileDownload / File: emd_16929.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 260 pix.
= 251.68 Å		0.97 Å/pix.
x 260 pix.
= 251.68 Å		0.97 Å/pix.
x 260 pix.
= 251.68 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.968 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.013
Minimum - Maximum-0.02084263 - 0.061483636
Average (Standard dev.)0.00023371997 (±0.003183333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 251.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16929_msk_1.map
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Half map: #2

Fileemd_16929_half_map_1.map
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Half map: #1

Fileemd_16929_half_map_2.map
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Sample components

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Entire : Transcription elongation complex with Spt4/5

EntireName: Transcription elongation complex with Spt4/5
Components
  • Complex: Transcription elongation complex with Spt4/5
    • Complex: Pyrococcus furiosus RNA polymerase
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo1N
      • Protein or peptide: DNA-directed RNA polymerase subunit beta
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo1C
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo3
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo7
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo4
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo5
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo6
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo11
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo10
      • Protein or peptide: DNA-directed RNA polymerase subunit Rpo12
    • Complex: Spt4/5
      • Protein or peptide: Transcription elongation factor Spt5
      • Protein or peptide: Transcription elongation factor Spt4
    • Complex: Nucleotide strands
      • DNA: DNA Template Strand
      • DNA: DNA Non-Template Strand
      • RNA: RNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Transcription elongation complex with Spt4/5

SupramoleculeName: Transcription elongation complex with Spt4/5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4, #15-#16, #5-#14

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Supramolecule #2: Pyrococcus furiosus RNA polymerase

SupramoleculeName: Pyrococcus furiosus RNA polymerase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4, #15-#16, #6, #8-#11
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)

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Supramolecule #3: Spt4/5

SupramoleculeName: Spt4/5 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5, #7
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)

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Supramolecule #4: Nucleotide strands

SupramoleculeName: Nucleotide strands / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #12-#14
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)

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Macromolecule #1: DNA-directed RNA polymerase subunit Rpo1N

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo1N / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 103.61225 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString: MQSMKKVIGS IEFGILSPQE IRKMSAVEVT VPDTYDDDGY PIEGGVMDKR MGVIDPGLRC ETCGGRAGEC PGHFGHIELA RPVIHVGFA KTIYRILEST CRECGRIKLT DEEIEEYMKK IELARNRRSE FNEIIKEIHK KAKERMVCPH CGAPQYPIKF E KPTIYWEI ...String:
MQSMKKVIGS IEFGILSPQE IRKMSAVEVT VPDTYDDDGY PIEGGVMDKR MGVIDPGLRC ETCGGRAGEC PGHFGHIELA RPVIHVGFA KTIYRILEST CRECGRIKLT DEEIEEYMKK IELARNRRSE FNEIIKEIHK KAKERMVCPH CGAPQYPIKF E KPTIYWEI RKDEQGNEYR HRLMPTEVRD WLEKIPDKDL PLLGLDPEKS RPEWMVLTVL PVPPVTARPS ITLETGIRAE DD LTHKLVD IIRINNRLKQ NIEAGAPQLI IEDLWDLLQY HVTTYINNEA PGVPPAKHKS GRPLKTLAQR LKGKEGRFRG NLS GKRVNF SARTVISPDP MISINEVGVP VEVAMELTVP EKVTEFNIER LRKMVLNGPD KYPGANYVID PEGRRRRIMD SNKE TLANQ LDIGWTVERH LMDGDIVLFN RQPSLHRMSI MAHRVRVMPY RTFRLNLAVC PPYNADFDGD EMNLHVPQTE EAQAE ARIL MEVQNHIISP RYGGPIIGGI QDHISGGYLL TREGAYFTRD EVEQMLMFAG VDITELPEPD KYDENGNPLW SGKTIF SLL LPEDLTVWYR NKLCDEPERC EALEKLIEEK LMPDPEEVRK LAYDGFVYIQ NGKLLSGAID KKAYGREDGI ILDLIVR EY GVERARQFLD QVTKLTIWVI THKGFTTGID DEDLPEEARD RIREIIREAE ERVNKLIEAY KRGELEPLPG KSLEDTLE S LIMAVLAEAR DNAGAVAEKY LGMDNHTVIM AKTGARGKIL NITQMAALLG QQSIRGKRLY RGFRGRVLSH FKPGDLGAR AKGFVVNSYK SGLTPQEYFF HAMGGREGLV DTAVRTAQSG YMQRRLINAL QDLKVEYDGT VRSPEGIIVQ FKYGEDGVDP MKSWRGKTV DVDRIIVRTL LKMRGNNKAK V

UniProtKB: DNA-directed RNA polymerase subunit Rpo1N

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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
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 127.188898 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString: MRGPTVVDVT PDDLWIVMES YWQEKGLVRQ HLDSYNAFIE RGLQEVVNEF GGIKPDIPDF EVKFGKIRVG EPEFQEPHGQ RKPLYPMDA RIRNLTYAAP LYLEMIPVIK GVEQDPVEVR IGELPIMLKS KVCRLYGLSD EELIKLGEDP KDPGGYFIIN G SERVIVSI ...String:
MRGPTVVDVT PDDLWIVMES YWQEKGLVRQ HLDSYNAFIE RGLQEVVNEF GGIKPDIPDF EVKFGKIRVG EPEFQEPHGQ RKPLYPMDA RIRNLTYAAP LYLEMIPVIK GVEQDPVEVR IGELPIMLKS KVCRLYGLSD EELIKLGEDP KDPGGYFIIN G SERVIVSI EDIAPNRTLV EKDERQEKYI AKVFSYRHGY RALVAVERRK DGILYVDIPN VPKPVKFVYV MRALGLERDK DI VEAVGND PEVQQIMFDN LEDASDITTQ QEALEFIGKL VAPGQAREYR LKRAEYVIDN NLLPHMGVSP EDRIKKAYYL GMM ALKVIE LSLGRRPEDD KDHYANKRLR LAGDLLKDLF RVAFSQLVKD IQYQMTKTYQ RKGDKYTFGN IHRFIRNSIR PDVL TERIE HALATGAWPG GRTGVSQLLD RTNYMSTLSH LRRVTSPLDR EQPHFEARDL HGTHWGRICP TETPEGPNCG LVKNL ALMA QITTAVPEEE IREYLMSLGI VPIEVRRPDP TLWRVYLNGV LIGTIEDGKA LVQRIRQDRR SGKISDVINV AYYEDV KEV YVNSDDGRVR RPLIIVENGV PKLTKEHVQA VKEGRLKWSD LIKMGIIEYL DAEEEENAYV AMWPWEVTEE HTHLELM PA AILGLPASLV PYPEHNAAPR NTYGAGMAKQ SLGLGWANFR IRVDTRGHLL HYPQIPLVNS RIMKAVGYED RPAGQNFV V AVLAYQGYNM EDAIIINKAS IERGLARSTF FRTYEAEEKK YLGGQTDKFE IPDPTVRGYR GEKYYRNLDE DGLIFPESK VEGKDVLVGR TSPPRFLEER GLGTVSLQER RETSVAVRPS EKGVVDKVII TETGDGTKLV KVTVRDLRIP ELGDKFASRH GQKGVIGLI VPQEDMPWTE SGIVPDLIVN PHGIPSRMTV GQLIEAIGGK VASLKGRRVD GTAFIGEPEE KLRKELEELG F KHTGREIM YDGITGKRLE ADIFIGVIYY QRLHHMVADK IHARSRGPVQ VLTKQPTEGR AREGGLRFGE MERDVLVGHG AA MLLIERL LEESDKTEVW VCENCGHIAL EDKRRRRVYC PVCGEEERIS KVEMSYAFKL LLDELKAMVI RPKLNLSERV

UniProtKB: DNA-directed RNA polymerase subunit beta

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Macromolecule #3: DNA-directed RNA polymerase subunit Rpo1C

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo1C / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 44.468156 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString: MVSLSTIKSL IEKKGANLPE NVKSELYEKL KKYNEKYKLT KAEIEAIIDD VVKEYERALV EPGEPVGTVA AQSIGEPSTQ MTLNTFHYA GVAEINVTLG LPRIIEIVDA RKNPSTPMMT VYLDEEHRYD RAKAEEVARR IEGTTLENLA RSTTLDLINF E FIVEIDPE ...String:
MVSLSTIKSL IEKKGANLPE NVKSELYEKL KKYNEKYKLT KAEIEAIIDD VVKEYERALV EPGEPVGTVA AQSIGEPSTQ MTLNTFHYA GVAEINVTLG LPRIIEIVDA RKNPSTPMMT VYLDEEHRYD RAKAEEVARR IEGTTLENLA RSTTLDLINF E FIVEIDPE RLERSGLTME KVVKKLESSF KSAEFEVDGY TLIVRPKKAD KISDLRRFAE KIKKHRLKGL SGVGKTIVRK EG DEYVIYT EGSNFKQVLK VPGVDPTRTR TNNIHEIAEV LGIEAARNAI IDEIVSTMQE QGLEVDIRHI MLVADMMTLD GIV RPIGRH GVVGEKSSVL ARAAFEITVQ HLFEAAEKGE VDNLNGVIEN VLIGQPVPVG TGMVKLTMKL PLRPQKEKEE V

UniProtKB: DNA-directed RNA polymerase subunit Rpo1C

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Macromolecule #4: DNA-directed RNA polymerase subunit Rpo3

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo3 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 31.688195 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString: MAGIEVQILE KKEDSIKFVL KGVHVSFANA LRRTILGEVP TFAVDEVEFY ENDSALFDEI IAHRLAMIPL TTPVDRFELD ALELDDYTV TLSLEAEGPG IVYSGDLKSD DPDVKPVNPN IPIVKLAEGQ RLVFNAYAKL GRGKDHAKWQ PGFVYYKYYT I VHISKSIP ...String:
MAGIEVQILE KKEDSIKFVL KGVHVSFANA LRRTILGEVP TFAVDEVEFY ENDSALFDEI IAHRLAMIPL TTPVDRFELD ALELDDYTV TLSLEAEGPG IVYSGDLKSD DPDVKPVNPN IPIVKLAEGQ RLVFNAYAKL GRGKDHAKWQ PGFVYYKYYT I VHISKSIP EWKELKKLAK KRGLPVEETE EEVLVTTIKP FYIPKDFEEY EGKEIWEEIV PNTYIFTVET NGELPVEEIV SI ALKILMR KADRFISELQ KLTSWSHPQF EKHHHHHH

UniProtKB: DNA-directed RNA polymerase subunit Rpo3

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Macromolecule #5: Transcription elongation factor Spt5

MacromoleculeName: Transcription elongation factor Spt5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 16.8166 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MAGKIFAVRV THGQEETTAK LIYSKVRTYN LPIYAILAPS RVKGYIFVEA PNKGVVDEAI RGIRHARGVL PGEVPFKEIE HFLEEKPAV SGLEPGDLVE VIAGPFKGQK AKVVKIDESK DEVVVQFIDA IVPIPVTIKG DYVRLISKLQ KEE

UniProtKB: Transcription elongation factor Spt5

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Macromolecule #6: DNA-directed RNA polymerase subunit Rpo5

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo5 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 9.260792 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString:
MAGKKEFSIF DHVLVPEHRV LSEEEKKALL EKYKITLAQL PQIKASDPAV KALGAKPGDV IEIKRKSPTA GVYYYYRVVV ED

UniProtKB: DNA-directed RNA polymerase subunit Rpo5

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Macromolecule #7: Transcription elongation factor Spt4

MacromoleculeName: Transcription elongation factor Spt4 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 6.965045 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSEKACRHCH YITSEDRCPV CGSRDLSEEW FDLVIIVDVE NSEIAKKIGA KVPGKYAIRV R

UniProtKB: Transcription elongation factor Spt4

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Macromolecule #8: DNA-directed RNA polymerase subunit Rpo6

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo6 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 6.24349 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString:
MFKYTRFEKA RIIGARALQI SMGAPVLIDV PPGITPLEAA ILEFEKGVIP ITVIRPS

UniProtKB: DNA-directed RNA polymerase subunit Rpo6

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Macromolecule #9: DNA-directed RNA polymerase subunit Rpo11

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo11 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 11.132764 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString:
MKIEVIKKEE NLLEFYLEGE DHTFANLLVE TLRENPHVKF TAYTIEHPIT MARKPRFRVV TDGEITPEEA LEEAAKKIFE RAKEVLEAW EKAVKS

UniProtKB: DNA-directed RNA polymerase subunit Rpo11

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Macromolecule #10: DNA-directed RNA polymerase subunit Rpo10

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo10 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 7.800158 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString:
MIVPVRCFTC GKVIGDKYYE FKRRVEAGED PEKVLDDLGL ERYCCRRMLL SHVELIDDIM HYRVY

UniProtKB: DNA-directed RNA polymerase subunit Rpo10

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Macromolecule #11: DNA-directed RNA polymerase subunit Rpo12

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo12 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 5.77005 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString:
MVEAVYRCFK CGREVKLDLS ITRDLRCPYC GSKILYKPRP KVPRRVKAI

UniProtKB: DNA-directed RNA polymerase subunit Rpo12

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Macromolecule #15: DNA-directed RNA polymerase subunit Rpo7

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo7 / type: protein_or_peptide / ID: 15 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 21.731455 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString:
MYKIVTVKDV VRIPPTMFTM DPKEAAKIIL RETYEGTYDK DEGVILSILE VKDIKDGIII PGDGATYHEV VFDVLVWEPK IHEVVEGYV ADVMPFGAFI RIGPIDGLVH ISQLMDDYVV YDERNKQFVG KEKKYLLKIG DLVRARIINI SAKSKVIREN R IGLTMRQP GLGKFEWIEK EKKKEKEEGK K

UniProtKB: DNA-directed RNA polymerase subunit Rpo7

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Macromolecule #16: DNA-directed RNA polymerase subunit Rpo4

MacromoleculeName: DNA-directed RNA polymerase subunit Rpo4 / type: protein_or_peptide / ID: 16 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 14.116311 KDa
Recombinant expressionOrganism: Pyrococcus furiosus DSM 3638 (archaea)
SequenceString:
MIGRGKLGEK YITIAEAKEL LLKRREEEVK AGIEEPLYYE ARLALEHAER FAKLPADKAK EAVEELMNAF EWMSDRIACK IVDIMPEDS MDLRVIFAKE EYQPTQEEMK QILDILDKYR E

UniProtKB: DNA-directed RNA polymerase subunit Rpo4

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Macromolecule #12: DNA Template Strand

MacromoleculeName: DNA Template Strand / type: dna / ID: 12 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 8.043188 KDa
SequenceString:
(DG)(DG)(DG)(DA)(DA)(DG)(DT)(DA)(DG)(DT) (DT)(DA)(DC)(DG)(DC)(DC)(DT)(DG)(DG)(DT) (DC)(DA)(DT)(DT)(DA)(DC)

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Macromolecule #13: DNA Non-Template Strand

MacromoleculeName: DNA Non-Template Strand / type: dna / ID: 13 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 7.93213 KDa
SequenceString:
(DG)(DT)(DA)(DA)(DT)(DG)(DA)(DC)(DC)(DA) (DG)(DG)(DC)(DG)(DT)(DA)(DA)(DC)(DT)(DA) (DC)(DT)(DT)(DC)(DC)(DC)

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Macromolecule #14: RNA

MacromoleculeName: RNA / type: rna / ID: 14 / Number of copies: 1
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 2.909823 KDa
SequenceString:
GACCAGGCG

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Macromolecule #17: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 17 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #18: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 18 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 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: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL CRYO ARM 200
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 22.0 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 52527
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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