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- PDB-8hr5: Cryo-EM structure of the CnCas12f1-sgRNA-DNA complex -

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Basic information

Entry
Database: PDB / ID: 8hr5
TitleCryo-EM structure of the CnCas12f1-sgRNA-DNA complex
Components
  • DNA (28-MER)
  • DNA (5'-D(*TP*AP*AP*CP*CP*TP*AP*AP*TP*AP*GP*AP*TP*GP*TP*GP*AP*A)-3')
  • Transposase
  • sgRNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / Complex / CRISPR-Cas / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


transposition / DNA recombination / DNA binding
Similarity search - Function
Transposase IS605, OrfB, C-terminal / Cas12f1-like, TNB domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Transposase
Similarity search - Component
Biological speciesClostridium novyi (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 3.73 Å
AuthorsLi, F. / Ji, Q.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21922705 China
National Natural Science Foundation of China (NSFC)2207783 China
National Natural Science Foundation of China (NSFC)91753127 China
CitationJournal: To Be Published
Title: Structure and engineering of miniature Clostridium novyi CRISPR-Cas12f1 with rare C-rich PAM specificity
Authors: Li, F. / Ji, Q.
History
DepositionDec 14, 2022Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transposase
B: Transposase
C: sgRNA
D: DNA (28-MER)
E: DNA (5'-D(*TP*AP*AP*CP*CP*TP*AP*AP*TP*AP*GP*AP*TP*GP*TP*GP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)206,8785
Polymers206,8785
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transposase / CnCas12f1


Mass: 58609.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium novyi (bacteria) / Gene: DFH04_07400 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A386YN77
#2: RNA chain sgRNA


Mass: 75498.406 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 DNA (28-MER)


Mass: 8612.591 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 DNA (5'-D(*TP*AP*AP*CP*CP*TP*AP*AP*TP*AP*GP*AP*TP*GP*TP*GP*AP*A)-3')


Mass: 5547.643 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: 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: Ternary complex of CnCas12f1-sgRNA-DNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Clostridium novyi (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO

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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: 1800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100719 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038918
ELECTRON MICROSCOPYf_angle_d0.60212554
ELECTRON MICROSCOPYf_dihedral_angle_d19.5822216
ELECTRON MICROSCOPYf_chiral_restr0.0381477
ELECTRON MICROSCOPYf_plane_restr0.0031139

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