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- PDB-8gkh: Structure of the Spizellomyces punctatus Fanzor (SpuFz) in comple... -

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Basic information

Entry
Database: PDB / ID: 8gkh
TitleStructure of the Spizellomyces punctatus Fanzor (SpuFz) in complex with omega RNA and target DNA
Components
  • DNA (35-MER)
  • DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')
  • Maltodextrin-binding protein (Fragment),OrfB_Zn_ribbon domain-containing protein
  • RNA (78-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / Fanzor / Eukaryotic / RNA-guided / nuclease / Gene editing / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


maltose binding / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing
Similarity search - Function
Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Maltodextrin-binding protein / Uncharacterized protein
Similarity search - Component
Biological speciesMethanosarcina mazei (archaea)
Spizellomyces punctatus (fungus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsXu, P. / Saito, M. / Zhang, F.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2023
Title: Fanzor is a eukaryotic programmable RNA-guided endonuclease.
Authors: Makoto Saito / Peiyu Xu / Guilhem Faure / Samantha Maguire / Soumya Kannan / Han Altae-Tran / Sam Vo / AnAn Desimone / Rhiannon K Macrae / Feng Zhang /
Abstract: RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both ...RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage. Although a few eukaryotic RNA-guided systems have been studied, including RNA interference and ribosomal RNA modification, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins, raising the possibility that eukaryotes are also equipped with CRISPR-Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life.
History
DepositionMar 19, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
N: DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')
P: Maltodextrin-binding protein (Fragment),OrfB_Zn_ribbon domain-containing protein
T: DNA (35-MER)
W: RNA (78-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,5287
Polymers158,4144
Non-polymers1143
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules NT

#1: DNA chain DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')


Mass: 4594.986 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (35-MER)


Mass: 10737.919 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules PW

#2: Protein Maltodextrin-binding protein (Fragment),OrfB_Zn_ribbon domain-containing protein


Mass: 118029.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanosarcina mazei (archaea), (gene. exp.) Spizellomyces punctatus (fungus)
Gene: malE / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A0F8NYV9, UniProt: A0A0L0H5U9
#4: RNA chain RNA (78-MER)


Mass: 25050.807 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spizellomyces punctatus (fungus) / Production host: Saccharomyces cerevisiae (brewer's yeast)

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Non-polymers , 3 types, 6 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O

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Details

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: SpuFz-wRNA-tgDNA complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Spizellomyces punctatus (fungus)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: 2600 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 51.41 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: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 501142 / Symmetry type: POINT

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