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- PDB-8qwf: ReChb - crRNA - target dsDNA complex in the presence of collatera... -

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Basic information

Entry
Database: PDB / ID: 8qwf
TitleReChb - crRNA - target dsDNA complex in the presence of collateral dsDNA
Components
  • ReChb
  • crRNA
  • non target DNA
  • target DNA
KeywordsBIOSYNTHETIC PROTEIN / Cas / cryo-EM / ancestral sequence reconstruction / biotechnology / DNA / RNA
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsLopez-Alonso, J.P. / Ubarretxena-Belandia, I. / Tascon, I.
Funding support1items
OrganizationGrant numberCountry
Other private
CitationJournal: Nat Biotechnol / Year: 2024
Title: A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.
Authors: Ylenia Jabalera / Igor Tascón / Sara Samperio / Jorge P López-Alonso / Monika Gonzalez-Lopez / Ana M Aransay / Guillermo Abascal-Palacios / Chase L Beisel / Iban Ubarretxena-Belandia / Raul Perez-Jimenez /
Abstract: The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs ...The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.
History
DepositionOct 19, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ReChb
C: crRNA
T: non target DNA
N: target DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)185,80410
Polymers185,6584
Non-polymers1466
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ReChb / alpha-synthetic Cas


Mass: 148954.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: RNA chain crRNA


Mass: 12696.498 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#3: DNA chain non target DNA


Mass: 11983.707 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#4: DNA chain target DNA


Mass: 12023.731 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Ctagcatatttatcctaaggcgttaccccaatgggaggg / Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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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: Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris1
22 mMMagnesium chlorideMgCl21
3150 mMSodium chlorideNaCl1
40.05 %CHAPS1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Details: 2s blotting

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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 magnification: 105000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.12 sec. / Electron dose: 50.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 34123
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPU2image acquisition
4cryoSPARC4.3.0CTF correction
7Cootmodel fitting
9cryoSPARC4.3.0initial Euler assignment
10cryoSPARC4.3.0final Euler assignment
11cryoSPARC4.3.0classification
12cryoSPARC3.4.03D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1526906
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 300509 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingDetails: ModelAngelo / Source name: Other / Type: other
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211882
ELECTRON MICROSCOPYf_angle_d0.49516325
ELECTRON MICROSCOPYf_dihedral_angle_d16.2282098
ELECTRON MICROSCOPYf_chiral_restr0.0381753
ELECTRON MICROSCOPYf_plane_restr0.0031839

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