EMDB-19033: Cryo-EM structure of Pyrococcus furiosus apo form RNA polymerase ...

Basic information

Entry

Database: EMDB / ID: EMD-19033

• Title: Cryo-EM structure of Pyrococcus furiosus apo form RNA polymerase contracted clamp conformation

Sample

• Complex: Pyrococcus furiosus apo form of RNA polymerase contracted clamp conformation
  • Protein or peptide: x 11 types
• Ligand: x 2 types

• Keywords: RNA / Polymerase / Elongation / Complex / Pyrococcus furiosus / Archaea / Transcription / DNA

Function / homology

Function:

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 / nucleotide binding / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm

Domain/homology:

DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo4 / HRDC domain superfamily / DNA-directed RNA polymerase, subunit E/RPC8 / S1 domain profile. / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / RNA polymerase subunit Rpb7-like / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / HRDC-like superfamily / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / 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 / DNA-directed RNA polymerase subunit Rpo11 / 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 Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RNA polymerases L / 13 to 16 Kd subunits signature. / 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, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / 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 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / Nucleic acid-binding, OB-fold

Component:

DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo12 / 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 / DNA-directed RNA polymerase subunit Rpo11

• Biological species: Pyrococcus furiosus DSM 3638 (archaea)
• Method: single particle reconstruction / cryo EM / Resolution: 3.02 Å
• Authors: Tarau DM / Reichelt R / Heiss FB / Pilsl M / Hausner W / Engel C / Grohmann D

Funding support

Germany, 1 items

Organization	Grant number	Country
German Research Foundation (DFG)	SFB960	Germany

• Citation: Journal: 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, 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

Deposition	Dec 4, 2023	-
Header (metadata) release	Apr 24, 2024	-
Map release	Apr 24, 2024	-
Update	May 15, 2024	-
Current status	May 15, 2024	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_19033.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 0.968 Å

Density

Contour Level	By AUTHOR: 0.095
Minimum - Maximum	-0.2534095 - 0.5410489
Average (Standard dev.)	0.00066239276 (±0.021771967)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 300 300 300 Spacing 300 300 300
Cell	A=B=C: 290.4 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_19033_msk_1.map

Half map: #2

• File: emd_19033_half_map_1.map

Half map: #1

• File: emd_19033_half_map_2.map

Sample components

Entire : Pyrococcus furiosus apo form of RNA polymerase contracted clamp c...

• Entire: Name: Pyrococcus furiosus apo form of RNA polymerase contracted clamp conformation

Components

• Complex: Pyrococcus furiosus apo form of RNA polymerase contracted clamp conformation
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo1NPolymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo1CPolymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo3Polymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo7Polymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo4Polymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo5Polymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo6Polymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo11Polymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo10Polymerase
  • Protein or peptide: DNA-directed RNA polymerase subunit Rpo12Polymerase
• Ligand: ZINC ION
• Ligand: MAGNESIUM ION

Supramolecule #1: Pyrococcus furiosus apo form of RNA polymerase contracted clamp c...

• Supramolecule: Name: Pyrococcus furiosus apo form of RNA polymerase contracted clamp conformation; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#11
• Source (natural): Organism: Pyrococcus furiosus DSM 3638 (archaea)

Macromolecule #1: DNA-directed RNA polymerase subunit Rpo1N

• Macromolecule: Name: 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 weight: Theoretical: 103.61225 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

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

Macromolecule #2: DNA-directed RNA polymerase subunit beta

• Macromolecule: Name: 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 weight: Theoretical: 127.188898 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

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

Macromolecule #3: DNA-directed RNA polymerase subunit Rpo1C

• Macromolecule: Name: 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 weight: Theoretical: 44.468156 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

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

Macromolecule #4: DNA-directed RNA polymerase subunit Rpo3

• Macromolecule: Name: 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 weight: Theoretical: 31.688195 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

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

Macromolecule #5: DNA-directed RNA polymerase subunit Rpo7

• Macromolecule: Name: DNA-directed RNA polymerase subunit Rpo7 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Pyrococcus furiosus DSM 3638 (archaea)
• Molecular weight: Theoretical: 21.731455 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

String:

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

Macromolecule #6: DNA-directed RNA polymerase subunit Rpo4

• Macromolecule: Name: DNA-directed RNA polymerase subunit Rpo4 / 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 weight: Theoretical: 14.116311 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

String:

MIGRGKLGEK YITIAEAKEL LLKRREEEVK AGIEEPLYYE ARLALEHAER FAKLPADKAK EAVEELMNAF EWMSDRIACK IVDIMPEDS MDLRVIFAKE EYQPTQEEMK QILDILDKYR E

UniProtKB: DNA-directed RNA polymerase subunit Rpo4

Macromolecule #7: DNA-directed RNA polymerase subunit Rpo5

• Macromolecule: Name: DNA-directed RNA polymerase subunit Rpo5 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
• Source (natural): Organism: Pyrococcus furiosus DSM 3638 (archaea)
• Molecular weight: Theoretical: 9.260792 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

String:

MAGKKEFSIF DHVLVPEHRV LSEEEKKALL EKYKITLAQL PQIKASDPAV KALGAKPGDV IEIKRKSPTA GVYYYYRVVV ED

UniProtKB: DNA-directed RNA polymerase subunit Rpo5

Macromolecule #8: DNA-directed RNA polymerase subunit Rpo6

• Macromolecule: Name: 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 weight: Theoretical: 6.24349 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

String:

MFKYTRFEKA RIIGARALQI SMGAPVLIDV PPGITPLEAA ILEFEKGVIP ITVIRPS

UniProtKB: DNA-directed RNA polymerase subunit Rpo6

Macromolecule #9: DNA-directed RNA polymerase subunit Rpo11

• Macromolecule: Name: 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 weight: Theoretical: 11.132764 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

String:

MKIEVIKKEE NLLEFYLEGE DHTFANLLVE TLRENPHVKF TAYTIEHPIT MARKPRFRVV TDGEITPEEA LEEAAKKIFE RAKEVLEAW EKAVKS

UniProtKB: DNA-directed RNA polymerase subunit Rpo11

Macromolecule #10: DNA-directed RNA polymerase subunit Rpo10

• Macromolecule: Name: 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 weight: Theoretical: 7.800158 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

String:

MIVPVRCFTC GKVIGDKYYE FKRRVEAGED PEKVLDDLGL ERYCCRRMLL SHVELIDDIM HYRVY

UniProtKB: DNA-directed RNA polymerase subunit Rpo10

Macromolecule #11: DNA-directed RNA polymerase subunit Rpo12

• Macromolecule: Name: 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 weight: Theoretical: 5.77005 KDa
• Recombinant expression: Organism: Pyrococcus furiosus DSM 3638 (archaea)

Sequence

String:

MVEAVYRCFK CGREVKLDLS ITRDLRCPYC GSKILYKPRP KVPRRVKAI

UniProtKB: DNA-directed RNA polymerase subunit Rpo12

Macromolecule #12: ZINC ION

• Macromolecule: Name: ZINC ION / type: ligand / ID: 12 / Number of copies: 5 / Formula: ZN
• Molecular weight: Theoretical: 65.409 Da

Macromolecule #13: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 13 / Number of copies: 1 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 8
• Vitrification: Cryogen name: ETHANE

Electron microscopy

• Microscope: JEOL CRYO ARM 200
• Electron beam: Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 1.0 e/Ų

Image processing

• Startup model: Type of model: OTHER
• Initial angle assignment: Type: ANGULAR RECONSTITUTION
• Final angle assignment: Type: ANGULAR RECONSTITUTION
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 111194