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- PDB-7wju: Cryo-EM structure of the AsCas12f1-sgRNAv1-dsDNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 7wju
TitleCryo-EM structure of the AsCas12f1-sgRNAv1-dsDNA ternary complex
Components
  • Non-target strand
  • OrfB_Zn_ribbon domain-containing protein
  • Target strand
  • sgRNAv1
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / targeting / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
Transposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endodeoxyribonuclease Cas12f1
Similarity search - Component
Biological speciesAcidibacillus sulfuroxidans (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsWu, Z. / Liu, D. / Shen, H. / Ji, Q.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21922705 China
National Natural Science Foundation of China (NSFC)91753127 China
National Natural Science Foundation of China (NSFC)2207783 China
CitationJournal: To Be Published
Title: Structure-directed functional evolution of the miniature CRISPR-AsCas12f1 system
Authors: Wu, Z. / Liu, D. / Pan, D. / Shen, H. / Ji, Q.
History
DepositionJan 7, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 18, 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: OrfB_Zn_ribbon domain-containing protein
B: OrfB_Zn_ribbon domain-containing protein
C: sgRNAv1
D: Target strand
E: Non-target strand


Theoretical massNumber of molelcules
Total (without water)170,1845
Polymers170,1845
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13790 Å2
ΔGint-80 kcal/mol
Surface area50940 Å2

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Components

#1: Protein OrfB_Zn_ribbon domain-containing protein


Mass: 48719.742 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidibacillus sulfuroxidans (bacteria) / Gene: BM613_13600 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2U3D0N8
#2: RNA chain sgRNAv1


Mass: 61643.578 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 Target strand


Mass: 9260.985 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 Non-target strand


Mass: 1840.227 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

Component
IDNameTypeEntity IDParent-IDSource
1AsCas12f1-sgRNAv1-dsDNA ternary complexCOMPLEXall0RECOMBINANT
2AsCas12f1COMPLEX#11RECOMBINANT
3sgRNA1COMPLEX#21RECOMBINANT
4dsDNADNACOMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Acidibacillus sulfuroxidans (bacteria)1765684
32synthetic construct (others)32630
43synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32synthetic construct (others)32630
43synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal defocus max: 1800 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 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: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1770878 / Symmetry type: POINT

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