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Yorodumi- EMDB-3981: Structure of mammalian RNA polymerase II elongation complex inhib... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3981 | ||||||||||||
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Title | Structure of mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin | ||||||||||||
Map data | Post-processed masked EM map of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin | ||||||||||||
Sample |
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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 ...: / 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 / organelle membrane / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / RNA polymerase II activity / transcription-coupled nucleotide-excision repair / transcription elongation by RNA polymerase I / tRNA transcription by RNA polymerase III / RNA polymerase I complex / RNA polymerase III complex / positive regulation of translational initiation / RNA polymerase II, core complex / core promoter sequence-specific DNA binding / translation initiation factor binding / transcription initiation at RNA polymerase II promoter / P-body / euchromatin / fibrillar center / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / single-stranded DNA binding / toxin activity / transcription by RNA polymerase II / nucleic acid binding / chromosome, telomeric region / single-stranded RNA binding / protein dimerization activity / nuclear speck / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / chromatin binding / nucleolus / DNA binding / zinc ion binding / nucleus / metal ion binding / cytosol Similarity search - Function | ||||||||||||
Biological species | Sus scrofa (pig) / synthetic construct (others) / Amanita phalloides (death cap) / Pig (pig) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.6 Å | ||||||||||||
Authors | Liu X / Farnung L / Wigge C / Cramer P | ||||||||||||
Funding support | Germany, 3 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3981.map.gz | 7.1 MB | EMDB map data format | |
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Header (meta data) | emd-3981-v30.xml emd-3981.xml | 38.9 KB 38.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_3981_fsc.xml | 9 KB | Display | FSC data file |
Images | emd_3981.png | 80.4 KB | ||
Masks | emd_3981_msk_1.map | 64 MB | Mask map | |
Others | emd_3981_half_map_1.map.gz emd_3981_half_map_2.map.gz | 49.6 MB 49.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3981 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3981 | HTTPS FTP |
-Validation report
Summary document | emd_3981_validation.pdf.gz | 431.2 KB | Display | EMDB validaton report |
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Full document | emd_3981_full_validation.pdf.gz | 430.3 KB | Display | |
Data in XML | emd_3981_validation.xml.gz | 15 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3981 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3981 | HTTPS FTP |
-Related structure data
Related structure data | 6exvMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3981.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Post-processed masked EM map of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_3981_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: Half-map post-refinement of mammalian RNA polymerase II elongation...
File | emd_3981_half_map_1.map | ||||||||||||
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Annotation | Half-map post-refinement of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map post-refinement of mammalian RNA polymerase II elongation...
File | emd_3981_half_map_2.map | ||||||||||||
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Annotation | Half-map post-refinement of mammalian RNA polymerase II elongation complex inhibited by %u03B1-amanitin | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Mammalian RNA polymerase II elongation complex inhibited by Alpha...
+Supramolecule #1: Mammalian RNA polymerase II elongation complex inhibited by Alpha...
+Supramolecule #2: RNA polymerase II elongation complex
+Supramolecule #3: Nucleic acids
+Supramolecule #4: Alpha-amanitin
+Macromolecule #1: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1
+Macromolecule #2: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2
+Macromolecule #3: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3
+Macromolecule #4: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3
+Macromolecule #5: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1
+Macromolecule #6: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1
+Macromolecule #7: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7
+Macromolecule #8: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC3
+Macromolecule #9: DNA-directed RNA polymerase II subunit RPB9
+Macromolecule #10: DNA-directed RNA polymerases I, II, and III subunit RPABC5
+Macromolecule #11: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4
+Macromolecule #12: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4
+Macromolecule #13: AMATOXIN
+Macromolecule #14: DNA (25-MER)
+Macromolecule #16: DNA (36-MER)
+Macromolecule #15: RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
+Macromolecule #17: ZINC ION
+Macromolecule #18: MAGNESIUM ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.244 mg/mL |
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Buffer | pH: 7.6 |
Grid | Model: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Alignment procedure | Coma free - Residual tilt: 10.0 mrad |
Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV |
Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 53.97 |
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Output model | PDB-6exv: |