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- EMDB-40984: 5TU-t1 - heterodimeric triplet polymerase ribozyme -

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Entry
Database: EMDB / ID: EMD-40984
Title5TU-t1 - heterodimeric triplet polymerase ribozyme
Map dataMap sharpened with deepEMhancer
Sample
  • Complex: 5TU-t1 - Triplet Polymerase Ribozyme
    • RNA: RNA (135-MER)
    • RNA: RNA (152-MER)
KeywordsPolymerase / Ribozyme / heterodimer / RNA
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsMcRae EKS / Kristoffersen E / Gallego I / Hansen K / Holliger P / Andersen ES
Funding support Denmark, Canada, United Kingdom, Germany, 8 items
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, 2023-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40984.png

Downloads & links

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Map

FileDownload / File: emd_40984.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened with deepEMhancer
Voxel sizeX=Y=Z: 1.29 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.0016844508 - 1.4878681
Average (Standard dev.)0.0007567582 (±0.018093262)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 268.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40984_msk_1.map
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AxesZYX

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Additional map: Unsharpened map

Fileemd_40984_additional_1.map
AnnotationUnsharpened map
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Half map: #1

Fileemd_40984_half_map_1.map
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Half map: #2

Fileemd_40984_half_map_2.map
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Sample components

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Entire : 5TU-t1 - Triplet Polymerase Ribozyme

EntireName: 5TU-t1 - Triplet Polymerase Ribozyme
Components
  • Complex: 5TU-t1 - Triplet Polymerase Ribozyme
    • RNA: RNA (135-MER)
    • RNA: RNA (152-MER)

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Supramolecule #1: 5TU-t1 - Triplet Polymerase Ribozyme

SupramoleculeName: 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.
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 92.7 KDa

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Macromolecule #1: RNA (135-MER)

MacromoleculeName: RNA (135-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 43.328703 KDa
SequenceString:
GACCAAUCUG CCCUCAGAGC UCGAGAACAU CUUCGGAUGC AGAGGAGGCA GGCUUCGGUG GCGCGAUAGC GCCAACGUCC UCAACCUCC AAUGCAUCCC ACCACAUGAU GAUGCCUGAA GAGCCUUGGU UUUUUG

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Macromolecule #2: RNA (152-MER)

MacromoleculeName: RNA (152-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 48.929094 KDa
SequenceString:
GGAUCUUCUC GAUCUAACAA AAAAGACAAA UCUGCCACAA AGCUUGAGAG CAUCUUCGGA UGCAGAGGCG GCAGCCUUCG GUGGCGCGA UAGCGCCAAC GUUCUCAACU AUGACACGCA AAACGCGUGC UCCGUUGAAU GGAGUUUAUC AUG

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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

Concentration3 mg/mL
BufferpH: 8
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: Local Refinement / Number images used: 86000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Avg.num./class: 40000
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel was initially built using DRRAFTER and then improved using ISOLDE in ChimeraX, Coot, Phenix RSR and QRNAS.
RefinementProtocol: AB INITIO MODEL
Output model

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

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