+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40984 | |||||||||||||||||||||||||||
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Title | 5TU-t1 - heterodimeric triplet polymerase ribozyme | |||||||||||||||||||||||||||
Map data | Map sharpened with deepEMhancer | |||||||||||||||||||||||||||
Sample |
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Keywords | Polymerase / Ribozyme / heterodimer / RNA | |||||||||||||||||||||||||||
Biological species | synthetic construct (others) | |||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.0 Å | |||||||||||||||||||||||||||
Authors | McRae EKS / Kristoffersen E / Gallego I / Hansen K / Holliger P / Andersen ES | |||||||||||||||||||||||||||
Funding support | Denmark, Canada, United Kingdom, Germany, 8 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2024 Title: Cryo-EM structure and functional landscape of an RNA polymerase ribozyme. Authors: Ewan K S McRae / Christopher J K Wan / Emil L Kristoffersen / Kalinka Hansen / Edoardo Gianni / Isaac Gallego / Joseph F Curran / James Attwater / Philipp Holliger / Ebbe S Andersen / Abstract: The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) - including a variant that uses trinucleotide ...The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) - including a variant that uses trinucleotide triphosphates (triplets) as substrates - have been created by in vitro evolution and are the closest functional analogues of the replicase, but the structural basis for their function is poorly understood. Here we use single-particle cryogenic electron microscopy (cryo-EM) and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The cryo-EM structure at 5-Å resolution reveals the TPR as an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit, resembling the shape of a left hand with thumb and fingers at a 70° angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme, whereby heterodimer formation and KL interactions preorganize the TPR for optimal primer-template duplex binding, triplet substrate discrimination, and templated RNA synthesis. These results provide a better understanding of TPR structure and function and should aid the engineering of more efficient PRs. | |||||||||||||||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_40984.map.gz | 30.8 MB | EMDB map data format | |
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Header (meta data) | emd-40984-v30.xml emd-40984.xml | 20.5 KB 20.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_40984_fsc.xml | 6.9 KB | Display | FSC data file |
Images | emd_40984.png | 35.5 KB | ||
Masks | emd_40984_msk_1.map | 34.3 MB | Mask map | |
Filedesc metadata | emd-40984.cif.gz | 5.5 KB | ||
Others | emd_40984_additional_1.map.gz emd_40984_half_map_1.map.gz emd_40984_half_map_2.map.gz | 17.1 MB 31.9 MB 31.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40984 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40984 | HTTPS FTP |
-Validation report
Summary document | emd_40984_validation.pdf.gz | 708.5 KB | Display | EMDB validaton report |
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Full document | emd_40984_full_validation.pdf.gz | 708.1 KB | Display | |
Data in XML | emd_40984_validation.xml.gz | 14 KB | Display | |
Data in CIF | emd_40984_validation.cif.gz | 18.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40984 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40984 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_40984.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Map sharpened with deepEMhancer | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.29 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_40984_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Unsharpened map
File | emd_40984_additional_1.map | ||||||||||||
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Annotation | Unsharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_40984_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_40984_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : 5TU-t1 - Triplet Polymerase Ribozyme
Entire | Name: 5TU-t1 - Triplet Polymerase Ribozyme |
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Components |
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-Supramolecule #1: 5TU-t1 - Triplet Polymerase Ribozyme
Supramolecule | Name: 5TU-t1 - Triplet Polymerase Ribozyme / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: Heterodimeric complex of two RNA strands comprising a functional triplet polymerase ribozyme. |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 92.7 KDa |
-Macromolecule #1: RNA (135-MER)
Macromolecule | Name: RNA (135-MER) / type: rna / ID: 1 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 43.328703 KDa |
Sequence | String: GACCAAUCUG CCCUCAGAGC UCGAGAACAU CUUCGGAUGC AGAGGAGGCA GGCUUCGGUG GCGCGAUAGC GCCAACGUCC UCAACCUCC AAUGCAUCCC ACCACAUGAU GAUGCCUGAA GAGCCUUGGU UUUUUG |
-Macromolecule #2: RNA (152-MER)
Macromolecule | Name: RNA (152-MER) / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 48.929094 KDa |
Sequence | String: GGAUCUUCUC GAUCUAACAA AAAAGACAAA UCUGCCACAA AGCUUGAGAG CAUCUUCGGA UGCAGAGGCG GCAGCCUUCG GUGGCGCGA UAGCGCCAAC GUUCUCAACU AUGACACGCA AAACGCGUGC UCCGUUGAAU GGAGUUUAUC AUG |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 3 mg/mL |
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Buffer | pH: 8 |
Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: 15mA on a GloQube Plus |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: LEICA EM GP |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Details | Model was initially built using DRRAFTER and then improved using ISOLDE in ChimeraX, Coot, Phenix RSR and QRNAS. |
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Refinement | Protocol: AB INITIO MODEL |
Output model | PDB-8t2p: |