+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0633 | |||||||||
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Title | RNA polymerase II elongation complex arrested at a CPD lesion | |||||||||
Map data | RNA polymerase II elongation complex stalled at a CPD lesion | |||||||||
Sample |
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Keywords | RNA polymerase / CPD / elongation complex / streptavidin grids / transcription / transferase-DNA-RNA complex | |||||||||
Function / homology | Function and homology information RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / : / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / termination of RNA polymerase II transcription / Formation of TC-NER Pre-Incision Complex / RNA polymerase II transcribes snRNA genes ...RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / : / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / termination of RNA polymerase II transcription / Formation of TC-NER Pre-Incision Complex / RNA polymerase II transcribes snRNA genes / RNA Polymerase I Promoter Escape / TP53 Regulates Transcription of DNA Repair Genes / Estrogen-dependent gene expression / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA-templated transcription / termination of RNA polymerase III transcription / RNA Polymerase II Pre-transcription Events / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / Dual incision in TC-NER / RNA polymerase I activity / termination of RNA polymerase I transcription / transcription initiation at RNA polymerase III promoter / tRNA transcription by RNA polymerase III / nucleolar large rRNA transcription by RNA polymerase I / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription initiation at RNA polymerase I promoter / transcription elongation by RNA polymerase I / RNA polymerase II activity / transcription-coupled nucleotide-excision repair / translesion synthesis / RNA polymerase I complex / transcription by RNA polymerase I / RNA polymerase III complex / transcription by RNA polymerase III / RNA polymerase II, core complex / transcription elongation by RNA polymerase II / transcription initiation at RNA polymerase II promoter / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / peroxisome / cytoplasmic stress granule / ribosome biogenesis / transcription by RNA polymerase II / nucleic acid binding / protein dimerization activity / mRNA binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytoplasm Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Lahiri I / Leshziner AE | |||||||||
Funding support | United States, 2 items
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Citation | Journal: J Struct Biol / Year: 2019 Title: 3.1 Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids. Authors: Indrajit Lahiri / Jun Xu / Bong Gyoon Han / Juntaek Oh / Dong Wang / Frank DiMaio / Andres E Leschziner / Abstract: Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact ...Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Å structure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)'s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface. | |||||||||
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_0633.map.gz | 124.6 MB | EMDB map data format | |
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Header (meta data) | emd-0633-v30.xml emd-0633.xml | 26.3 KB 26.3 KB | Display Display | EMDB header |
Images | emd_0633.png | 201.6 KB | ||
Filedesc metadata | emd-0633.cif.gz | 9.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0633 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0633 | HTTPS FTP |
-Related structure data
Related structure data | 6o6cMC 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_0633.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | RNA polymerase II elongation complex stalled at a CPD lesion | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.16 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
+Entire : RNA polymerase II elongation complex stalled at a CPD lesion
+Supramolecule #1: RNA polymerase II elongation complex stalled at a CPD lesion
+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 polymerases I, II, and III subunit RPABC1
+Macromolecule #5: DNA-directed RNA polymerases I, II, and III subunit RPABC2
+Macromolecule #6: DNA-directed RNA polymerases I, II, and III subunit RPABC3
+Macromolecule #7: DNA-directed RNA polymerase II subunit RPB9
+Macromolecule #8: DNA-directed RNA polymerases I, II, and III subunit RPABC5
+Macromolecule #9: DNA-directed RNA polymerase II subunit RPB11
+Macromolecule #10: DNA-directed RNA polymerases I, II, and III subunit RPABC4
+Macromolecule #11: RNA (5'-R(P*AP*UP*CP*GP*AP*GP*AP*GP*G)-3')
+Macromolecule #12: DNA (5'-D(P*GP*GP*AP*GP*AP*AP*GP*GP*AP*GP*CP*AP*GP*AP*GP*C)-3')
+Macromolecule #13: DNA (27-MER)
+Macromolecule #14: ZINC ION
+Macromolecule #15: MAGNESIUM ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: Quantifoil R2/2 / Material: GOLD / Mesh: 300 Details: The grids had a monolayer of streptavidin crystals on them. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV Details: The sample was manually wicked from the grid. 1.2 uL sample buffer was then applied to the streptavidin side. The grid was then blotted.. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 6.0 sec. / Average electron dose: 51.7 e/Å2 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Initial angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0) |
Final angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0) |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 61654 |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
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Output model | PDB-6o6c: |