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- EMDB-3981: Structure of mammalian RNA polymerase II elongation complex inhib... -

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Basic information

Entry
Database: EMDB / ID: EMD-3981
TitleStructure of mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
Map dataPost-processed masked EM map of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin
Sample
  • Complex: Mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
    • Complex: RNA polymerase II elongation complex
      • Protein or peptide: x 12 types
    • Complex: Nucleic acidsNucleic acid
      • DNA: x 2 types
      • RNA: x 1 types
    • Complex: Alpha-amanitinΑ-Amanitin
      • Protein or peptide: x 1 types
  • Ligand: x 2 types
Function / homology
Function and homology information


B-WICH complex positively regulates rRNA expression / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase I Transcription Termination / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase III Transcription Initiation From Type 3 Promoter / Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs ...B-WICH complex positively regulates rRNA expression / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase I Transcription Termination / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase III Transcription Initiation From Type 3 Promoter / Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs / RNA Polymerase II Pre-transcription Events / TP53 Regulates Transcription of DNA Repair Genes / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Major Pathway / mRNA Splicing - Minor Pathway / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Elongation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Pol II CTD phosphorylation and interaction with CE / Estrogen-dependent gene expression / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / : / : / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / positive regulation of translational initiation / RNA polymerase II activity / organelle membrane / transcription-coupled nucleotide-excision repair / RNA polymerase I complex / transcription by RNA polymerase I / RNA polymerase III complex / transcription by RNA polymerase III / RNA polymerase II, core complex / translation initiation factor binding / core promoter sequence-specific DNA binding / P-body / transcription initiation at RNA polymerase II promoter / euchromatin / ribonucleoside binding / fibrillar center / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / single-stranded DNA binding / toxin activity / transcription by RNA polymerase II / single-stranded RNA binding / chromosome, telomeric region / nucleic acid binding / protein dimerization activity / nuclear speck / nucleotide binding / DNA-templated transcription / chromatin binding / nucleolus / DNA binding / zinc ion binding / metal ion binding / nucleus / cytosol
Similarity search - Function
RNA-binding domain, S1 / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 ...RNA-binding domain, S1 / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Rpb4/RPC9 superfamily / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Zinc finger TFIIS-type signature. / RNA polymerase subunit Rpb7-like / HRDC-like superfamily / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA-directed RNA polymerase subunit Rpo11 / 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 Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RNA polymerases L / 13 to 16 Kd subunits signature. / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / 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 Rpb6 / 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
Similarity search - Domain/homology
RNA polymerase II subunit D / DNA-directed RNA polymerase II subunit RPB11-a / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase II subunit RPB7 / RNA polymerase II, I and III subunit K / DNA-directed RNA polymerase II subunit E ...RNA polymerase II subunit D / DNA-directed RNA polymerase II subunit RPB11-a / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase II subunit RPB7 / RNA polymerase II, I and III subunit K / DNA-directed RNA polymerase II subunit E / DNA-directed RNA polymerase II subunit RPB9 / Alpha-amanitin proprotein
Similarity search - Component
Biological speciesSus scrofa (pig) / synthetic construct (others) / Amanita phalloides (death cap) / Pig (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsLiu X / Farnung L / Wigge C / Cramer P
Funding support Germany, 3 items
OrganizationGrant numberCountry
European Research Council693023 Germany
German Research FoundationSPP1935 Germany
German Research FoundationSFB860 Germany
CitationJournal: J Biol Chem / Year: 2018
Title: Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by α-amanitin.
Authors: Xiangyang Liu / Lucas Farnung / Christoph Wigge / Patrick Cramer /
Abstract: RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step ...RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.
History
DepositionNov 9, 2017-
Header (metadata) releaseDec 20, 2017-
Map releaseMar 21, 2018-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0577
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0577
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6exv
  • Surface level: 0.0577
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3981.png

Downloads & links

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Map

FileDownload / File: emd_3981.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed masked EM map of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0577 / Movie #1: 0.0577
Minimum - Maximum-0.30933884 - 0.446918
Average (Standard dev.)0.00072788406 (±0.012988935)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.3090.4470.001

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Supplemental data

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Mask #1

Fileemd_3981_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map post-refinement of mammalian RNA polymerase II elongation...

Fileemd_3981_half_map_1.map
AnnotationHalf-map post-refinement of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map post-refinement of mammalian RNA polymerase II elongation...

Fileemd_3981_half_map_2.map
AnnotationHalf-map post-refinement of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mammalian RNA polymerase II elongation complex inhibited by Alpha...

EntireName: Mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
Components
  • Complex: Mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
    • Complex: RNA polymerase II elongation complex
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1Polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2Polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3Polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3Polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1RNA polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1RNA polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7Polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC3RNA polymerase
      • Protein or peptide: DNA-directed RNA polymerase II subunit RPB9Polymerase
      • Protein or peptide: DNA-directed RNA polymerases I, II, and III subunit RPABC5RNA polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4RNA polymerase
      • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4RNA polymerase
    • Complex: Nucleic acidsNucleic acid
      • DNA: DNA (25-MER)
      • RNA: RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
      • DNA: DNA (36-MER)
    • Complex: Alpha-amanitinΑ-Amanitin
      • Protein or peptide: AMATOXIN
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Mammalian RNA polymerase II elongation complex inhibited by Alpha...

SupramoleculeName: Mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#16
Molecular weightTheoretical: 607 KDa

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Supramolecule #2: RNA polymerase II elongation complex

SupramoleculeName: RNA polymerase II elongation complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#12
Source (natural)Organism: Sus scrofa (pig) / Tissue: Thymus

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Supramolecule #3: Nucleic acids

SupramoleculeName: Nucleic acids / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #14-#16
Source (natural)Organism: synthetic construct (others)

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Supramolecule #4: Alpha-amanitin

SupramoleculeName: Alpha-amanitin / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #13
Source (natural)Organism: Amanita phalloides (death cap)

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Macromolecule #1: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 217.450078 KDa
SequenceString: MHGGGPPSGD SACPLRTIKR VQFGVLSPDE LKRMSVTEGG IKYPETTEGG RPKLGGLMDP RQGVIERTGR CQTCAGNMTE CPGHFGHIE LAKPVFHVGF LVKTMKVLRC VCFFCSKLLV DSNNPKIKDI LAKSKGQPKK RLTHVYDLCK GKNICEGGEE M DNKFGVEQ ...String:
MHGGGPPSGD SACPLRTIKR VQFGVLSPDE LKRMSVTEGG IKYPETTEGG RPKLGGLMDP RQGVIERTGR CQTCAGNMTE CPGHFGHIE LAKPVFHVGF LVKTMKVLRC VCFFCSKLLV DSNNPKIKDI LAKSKGQPKK RLTHVYDLCK GKNICEGGEE M DNKFGVEQ PEGDEDLTKE KGHGGCGRYQ PRIRRSGLEL YAEWKHVNED SQEKKILLSP ERVHEIFKRI SDEECFVLGM EP RYARPEW MIVTVLPVPP LSVRPAVVMQ GSARNQDDLT HKLADIVKIN NQLRRNEQNG AAAHVIAEDV KLLQFHVATM VDN ELPGLP RAMQKSGRPL KSLKQRLKGK EGRVRGNLMG KRVDFSARTV ITPDPNLSID QVGVPRSIAA NMTFAEIVTP FNID RLQEL VRRGNSQYPG AKYIIRDNGD RIDLRFHPKP SDLHLQTGYK VERHMCDGDI VIFNRQPTLH KMSMMGHRVR ILPWS TFRL NLSVTTPYNA DFDGDEMNLH LPQSLETRAE IQELAMVPRM IVTPQSNRPV MGIVQDTLTA VRKFTKRDVF LERGEV MNL LMFLSTWDGK VPQPAILKPR PLWTGKQIFS LIIPGHINCI RTHSTHPDDE DSGPYKHISP GDTKVVVENG ELIMGIL CK KSLGTSAGSL VHISYLEMGH DITRLFYSNI QTVINNWLLI EGHTIGIGDS IADSKTYQDI QNTIKKAKQD VIEVIEKA H NNELEPTPGN TLRQTFENQV NRILNDARDK TGSSAQKSLS EYNNFKSMVV SGAKGSKINI SQVIAVVGQQ NVEGKRIPF GFKHRTLPHF IKDDYGPESR GFVENSYLAG LTPTEFFFHA MGGREGLIDT AVKTAETGYI QRRLIKSMES VMVKYDATVR NSINQVVQL RYGEDGLAGE SVEFQNLATL KPSNKAFEKK FRFDYTNERA LRRTLQEDLV KDVLSNAHIQ NELEREFERM R EDREVLRV IFPTGDSKVV LPCNLLRMIW NAQKIFHINP RLPSDLHPIK VVEGVKELSK KLVIVNGDDP LSRQAQENAT LL FNIHLRS TLCSRRMAEE FRLSGEAFDW LLGEIESKFN QAIAHPGEMV GALAAQSLGE PATQMTLNTF HYAGVSAKNV TLG VPRLKE LINISKKPKT PSLTVFLLGQ SARDAERAKD ILCRLEHTTL RKVTANTAIY YDPNPQSTVV AEDQEWVNVY YEMP DFDVA RISPWLLRVE LDRKHMTDRK LTMEQIAEKI NAGFGDDLNC IFNDDNAEKL VLRIRIMNSD ENKMQEEEEV VDKMD DDVF LRCIESNMLT DMTLQGIEQI SKVYMHLPQT DNKKKIIITE DGEFKALQEW ILETDGVSLM RVLSEKDVDP VRTTSN DIV EIFTVLGIEA VRKALERELY HVISFDGSYV NYRHLALLCD TMTCRGHLMA ITRHGVNRQD TGPLMKCSFE ETVDVLM EA AAHGESDPMK GVSENIMLGQ LAPAGTGCFD LLLDAEKCKY GMEIPTNIPG LGAAGPTGMF FGSAPSPMGG ISPAMTPW N QGATPAYGAW SPSVGSGMTP GAAGFSPSAA SDASGFSPGY SPAWSPTPGS PGSPGPSSPY IPSPGGAMSP SYSPTSPAY EPRSPGGYTP QSPSYSPTSP SYSPTSPSYS PTSPNYSPTS PSYSPTSPSY SPTSPSYSPT SPSYSPTSPS YSPTSPSYSP TSPSYSPTS PSYSPTSPSY SPTSPSYSPT SPSYSPTSPS YSPTSPSYSP TSPSYSPTSP SYSPTSPNYS PTSPNYTPTS P SYSPTSPS YSPTSPNYTP TSPNYSPTSP SYSPTSPSYS PTSPSYSPSS PRYTPQSPTY TPSSPSYSPS SPSYSPTSPK YT PTSPSYS PSSPEYTPTS PKYSPTSPKY SPTSPKYSPT SPTYSPTTPK YSPTSPTYSP TSPVYTPTSP KYSPTSPTYS PTS PKYSPT SPTYSPTSPK GSTYSPTSPG YSPTSPTYSL TSPAISPDDS DEEN

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Macromolecule #2: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 133.201625 KDa
SequenceString: MQYDEDDDEI TPDLWQEACW IVISSYFDEK GLVRQQLDSF DEFIQMSVQR IVEDAPPIDL QAEAQHASGE VEEPPRYLLK FEQIYLSKP THWERDGAPS PMMPNEARLR NLTYSAPLYV DITKTVIKEG EEQLQTQHQK TFIGKIPIML RSTYCLLNGL T DRDLCELN ...String:
MQYDEDDDEI TPDLWQEACW IVISSYFDEK GLVRQQLDSF DEFIQMSVQR IVEDAPPIDL QAEAQHASGE VEEPPRYLLK FEQIYLSKP THWERDGAPS PMMPNEARLR NLTYSAPLYV DITKTVIKEG EEQLQTQHQK TFIGKIPIML RSTYCLLNGL T DRDLCELN ECPLDPGGYF IINGSEKVLI AQEKMATNTV YVFAKKDSKY AYTGECRSCL ENSSRPTSTI WVSMLARGGQ GA KKSAIGQ RIVATLPYIK QEVPIIIVFR ALGFVSDRDI LEHIIYDFED PEMMEMVKPS LDEAFVIQEQ NVALNFIGSR GAK PGVTKE KRIKYAKEVL QKEMLPHVGV SDFCETKKAY FLGYMVHRLL LAALGRRELD DRDHYGNKRL DLAGPLLAFL FRGM FKNLL KEVRIYAQKF IDRGKDFNLE LAIKTRIISD GLKYSLATGN WGDQKKAHQA RAGVSQVLNR LTFASTLSHL RRLNS PIGR DGKLAKPRQL HNTLWGMVCP AETPEGHAVG LVKNLALMAY ISVGSQPSPI LEFLEEWSME NLEEISPAAI ADATKI FVN GCWVGIHKDP EQLMNTLRKL RRQMDIIVSE VSMIRDIRER EIRIYTDAGR ICRPLLIVEK QKLLLKKRHI DQLKERE YN NYSWQDLVAS GVVEYIDTLE EETVMLAMTP DDLQEKEVAY CSTYTHCEIH PSMILGVCAS IIPFPDHNQS PRNTYQSA M GKQAMGVYIT NFHVRMDTLA HVLYYPQKPL VTTRSMEYLR FRELPAGINS IVAIASYTGY NQEDSVIMNR SAVDRGFFR SVFYRSYKEQ ESKKGFDQEE VFEKPTRETC QGMRHAIYDK LDDDGLIAPG VRVSGDDVII GKTVTLPENE DELEGTNRRY TKRDCSTFL RTSETGIVDQ VMVTLNQEGY KFCKIRVRSV RIPQIGDKFA SRHGQKGTCG IQYRQEDMPF TCEGITPDII I NPHAIPSR MTIGHLIECL QGKVSANKGE IGDATPFNDA VNVQKISNLL SDYGYHLRGN EVLYNGFTGR KITSQIFIGP TY YQRLKHM VDDKIHSRAR GPIQILNRQP MEGRSRDGGL RFGEMERDCQ IAHGAAQFLR ERLFEASDPY QVHVCNLCGI MAI ANTRTH TYECRGCRNK TQISLVRMPY ACKLLFQELM SMSIAPRMMS V

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Macromolecule #3: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 31.439074 KDa
SequenceString: MPYANQPTVR ITELTDENVK FIIENTDLAV ANSIRRVFIA EVPIIAIDWV QIDANSSVLH DEFIAHRLGL IPLTSDDIVD KLQYSRDCT CEEFCPECSV EFTLDVRCNE DQTRHVTSRD LISNSPRVIP VTSRNRDNDP SDYVEQDDIL IVKLRKGQEL R LRAYAKKG ...String:
MPYANQPTVR ITELTDENVK FIIENTDLAV ANSIRRVFIA EVPIIAIDWV QIDANSSVLH DEFIAHRLGL IPLTSDDIVD KLQYSRDCT CEEFCPECSV EFTLDVRCNE DQTRHVTSRD LISNSPRVIP VTSRNRDNDP SDYVEQDDIL IVKLRKGQEL R LRAYAKKG FGKEHAKWNP TAGVAFEYDP DNALRHTVYP KPEEWPKSEY SELDEDESQA PYDPNGKPER FYYNVESCGS LR PETIVLS ALSGLKKKLS DLQTQLSHEI QSDVLTIN

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Macromolecule #4: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 16.331255 KDa
SequenceString:
MAAGGSDPRA GDVEEDASQL IFPKEFETAE TLLNSEVHML LEHRKQQNES AEDEQELSEV FMKTLNYTAR FSRFKNRETI ASVRSLLLQ KKLHKFELAC LANLCPETAE ESKALIPSLE GRFEDEELQQ ILDDIQTKRS FQY

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Macromolecule #5: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1
type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 24.644318 KDa
SequenceString: MDDEEETYRL WKIRKTIMQL CHDRGYLVTQ DELDQTLEEF KAQFGDKPSE GRPRRTDLTV LVAHNDDPTD QMFVFFPEEP KVGIKTIKV YCQRMQEENI TRALIVVQQG MTPSAKQSLV DMAPKYILEQ FLQQELLINI TEHELVPEHV VMTKEEVTEL L ARYKLREN ...String:
MDDEEETYRL WKIRKTIMQL CHDRGYLVTQ DELDQTLEEF KAQFGDKPSE GRPRRTDLTV LVAHNDDPTD QMFVFFPEEP KVGIKTIKV YCQRMQEENI TRALIVVQQG MTPSAKQSLV DMAPKYILEQ FLQQELLINI TEHELVPEHV VMTKEEVTEL L ARYKLREN QLPRIQAGDP VARYFGIKRG QVVKIIRPSE TAGRYITYRL VQ

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Macromolecule #6: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1
type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 14.477001 KDa
SequenceString:
MSDNEDNFDG DDFDDVEEDE GLDDLENAEE EGQENVEILP SGERPQANQK RITTPYMTKY ERARVLGTRA LQIAMCAPVM VELEGETDP LLIAMKELKA RKIPIIIRRY LPDGSYEDWG VDELIISD

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Macromolecule #7: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 19.314283 KDa
SequenceString:
MFYHISLEHE ILLHPRYFGP NLLNTVKQKL FTEVEGTCTG KYGFVIAVTT IDNIGAGVIQ PGRGFVLYPV KYKAIVFRPF KGEVVDAVV TQVNKVGLFT EIGPMSCFIS RHSIPSEMEF DPNSNPPCYK TMDEDIVIQQ DDEIRLKIVG TRVDKNDIFA I GSLMDDYL GLVS

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Macromolecule #8: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC3

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC3
type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 17.162273 KDa
SequenceString:
MAGILFEDIF DVKDIDPEGK KFDRVSRLHC ESESFKMDLI LDVNIQIYPV DLGDKFRLVI ASTLYEDGTL DDGEYNPTDD RPSRADQFE YVMYGKVYRI EGDETSTEAA TRLSAYVSYG GLLMRLQGDA NNLHGFEVDS RVYLLMKKLA F

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Macromolecule #9: DNA-directed RNA polymerase II subunit RPB9

MacromoleculeName: DNA-directed RNA polymerase II subunit RPB9 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 14.541221 KDa
SequenceString:
MEPDGTYEPG FVGIRFCQEC NNMLYPKEDK ENRILLYACR NCDYQQEADN SCIYVNKITH EVDELTQIIA DVSQDPTLPR TEDHPCQKC GHKEAVFFQS HSARAEDAMR LYYVCTAPHC GHRWTE

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Macromolecule #10: DNA-directed RNA polymerases I, II, and III subunit RPABC5

MacromoleculeName: DNA-directed RNA polymerases I, II, and III subunit RPABC5
type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 7.655123 KDa
SequenceString:
MIIPVRCFTC GKIVGNKWEA YLGLLQAEYT EGDALDALGL KRYCCRRMLL AHVDLIEKLL NYAPLEK

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Macromolecule #11: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4
type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 13.310284 KDa
SequenceString:
MNAPPAFESF LLFEGEKKIT INKDTKVPNA CLFTINKEDH TLGNIIKSQL LKDPQVLFAG YKVPHPLEHK IIIRVQTTPD YSPQEAFTN AITDLISELS LLEERFRVAI KDKQEGIE

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Macromolecule #12: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4
type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: Thymus
Molecular weightTheoretical: 7.018244 KDa
SequenceString:
MDTQKDVQPP KQQPMIYICG ECHTENEIKS RDPIRCRECG YRIMYKKRTK RLVVFDAR

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Macromolecule #13: AMATOXIN

MacromoleculeName: AMATOXIN / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Amanita phalloides (death cap)
Molecular weightTheoretical: 939.004 Da
SequenceString:
(ILX)(TRX)GIG(CSX)N(HYP)

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Macromolecule #14: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 14 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.303563 KDa
SequenceString:
(DG)(DG)(DC)(DA)(DG)(DT)(DA)(DC)(DT)(DA) (DG)(DT)(DA)(DT)(DT)(DC)(DT)(DA)(DG)(DT) (DA)(DT)(DT)(DG)(DA)(DA)(DA)(DG)(DT) (DA)(DC)(DT)(DT)(DG)(DA)(DG)(DC)(DT)(DT) (DG) (DA)(DT)(DC)

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Macromolecule #16: DNA (36-MER)

MacromoleculeName: DNA (36-MER) / type: dna / ID: 16 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.178483 KDa
SequenceString:
(DG)(DA)(DT)(DC)(DA)(DA)(DG)(DC)(DT)(DC) (DA)(DA)(DG)(DT)(DA)(DC)(DT)(DT)(DA)(DA) (DG)(DC)(DC)(DT)(DG)(DG)(DT)(DC)(DT) (DA)(DT)(DA)(DC)(DT)(DA)(DG)(DT)(DA)(DC) (DT) (DG)(DC)(DC)

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Macromolecule #15: RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')

MacromoleculeName: RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
type: rna / ID: 15 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.414902 KDa
SequenceString:
UAUAUGCAUA AAGACCAGGC

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Macromolecule #17: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 17 / Number of copies: 8 / 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

Concentration0.244 mg/mL
BufferpH: 7.6
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Alignment procedureComa free - Residual tilt: 10.0 mrad
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Number grids imaged: 1 / Number real images: 2264 / Average exposure time: 10.0 sec. / Average electron dose: 35.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 207410
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

3219

Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 1
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 1
Projection matching processing - Angular sampling: 1.8 degrees
Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 134512
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 53.97
Output model

PDB-6exv:
Structure of mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin

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