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- PDB-8t2p: 5TU-t1 - heterodimeric triplet polymerase ribozyme -

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Basic information

Entry
Database: PDB / ID: 8t2p
Title5TU-t1 - heterodimeric triplet polymerase ribozyme
Components
  • RNA (135-MER)
  • RNA (152-MER)
KeywordsRNA / Polymerase / Ribozyme / heterodimer
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5 Å
AuthorsMcRae, E.K.S. / Kristoffersen, E. / Gallego, I. / Hansen, K. / Holliger, P. / Andersen, E.S.
Funding support Denmark, Canada, United Kingdom, Germany, 8items
OrganizationGrant numberCountry
Danish Council for Independent Research9040-00425B Denmark
Novo Nordisk FoundationNNF21OC0070452 Denmark
Natural Sciences and Engineering Research Council (NSERC, Canada)532417 Canada
The Carlsberg FoundationCF20-0635 Denmark
LundbeckfondenR250-2017-1502 Denmark
UK Research and Innovation (UKRI)MC_U105178804 United Kingdom
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUNDH2020-MSCA-IF-2018-845303 Germany
The Carlsberg FoundationCF17-0809 Denmark
CitationJournal: 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
DepositionJun 6, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA (135-MER)
B: RNA (152-MER)


Theoretical massNumber of molelcules
Total (without water)92,2582
Polymers92,2582
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (135-MER)


Mass: 43328.703 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: RNA chain RNA (152-MER)


Mass: 48929.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 5TU-t1 - Triplet Polymerase Ribozyme / Type: COMPLEX
Details: Heterodimeric complex of two RNA strands comprising a functional triplet polymerase ribozyme.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.0927 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 8
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15mA on a GloQube Plus / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategoryDetails
2EPUimage acquisition
4cryoSPARCCTF correction
7PHENIXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstructionLocal Refinement
13Rosettamodel refinement
14ISOLDEmodel refinement
15UCSF ChimeraXmodel refinement
16Cootmodel refinement
17PHENIXmodel refinement
18Ambermodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Details: Model was initially built using DRRAFTER and then improved using ISOLDE in ChimeraX, Coot, Phenix RSR and QRNAS.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0066828
ELECTRON MICROSCOPYf_angle_d0.79710637
ELECTRON MICROSCOPYf_dihedral_angle_d17.863438
ELECTRON MICROSCOPYf_chiral_restr0.0521435
ELECTRON MICROSCOPYf_plane_restr0.005287

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