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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: Nature Biotechnology / Year: 2024
Title: A resurrected ancestor of Cas12a expands target access and substrate 2 recognition for nucleic-acid editing and detection
Authors: Jabalera, Y. / Tascon, I. / Samperio, S. / Lopez-Alonso, J.P. / Gonzalez-Lopez, M. / Aransay, A.M. / Abascal-Palacios, G. / Beisel, C.L. / Ubarretxena-Belandia, I. / Jimenez-Perez, R.
History
DepositionOct 19, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 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: 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

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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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