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- PDB-9cev: Spizellomyces punctatus Fanzor (SpuFz) State 2 -

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Basic information

Entry
Database: PDB / ID: 9cev
TitleSpizellomyces punctatus Fanzor (SpuFz) State 2
Components
  • DNA (35-MER)
  • DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')
  • Maltose/maltodextrin-binding periplasmic protein,Spizellomyces punctatus Fanzor 1
  • RNA (81-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / Fanzor / Eukaryotic / RNA-guided / nuclease / Gene editing / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / cell chemotaxis ...detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / outer membrane-bounded periplasmic space / periplasmic space / DNA damage response / membrane
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) / Uncharacterized protein / Maltose/maltodextrin-binding periplasmic protein
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Spizellomyces punctatus (fungus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsXu, P. / Saito, M. / Zhang, F.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Cell / Year: 2024
Title: Structural insights into the diversity and DNA cleavage mechanism of Fanzor.
Authors: Peiyu Xu / Makoto Saito / Guilhem Faure / Samantha Maguire / Samuel Chau-Duy-Tam Vo / Max E Wilkinson / Huihui Kuang / Bing Wang / William J Rice / Rhiannon K Macrae / Feng Zhang /
Abstract: Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different ...Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.
History
DepositionJun 27, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 2.0Sep 25, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Non-polymer description / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / chem_comp_atom / chem_comp_bond / em_admin / em_software / entity / entity_src_gen / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_residues / pdbx_validate_close_contact / pdbx_validate_rmsd_angle / pdbx_validate_rmsd_bond / pdbx_validate_torsion / refine / refine_ls_restr / struct_asym / struct_conf / struct_conn / struct_ref_seq / struct_ref_seq_dif / struct_sheet_range
Item: _chem_comp.formula / _chem_comp.formula_weight ..._chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _em_admin.last_update / _entity_src_gen.pdbx_gene_src_scientific_name / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq_dif.pdbx_auth_seq_num / _struct_sheet_range.beg_auth_comp_id / _struct_sheet_range.beg_auth_seq_id / _struct_sheet_range.beg_label_comp_id / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_auth_comp_id / _struct_sheet_range.end_auth_seq_id / _struct_sheet_range.end_label_comp_id / _struct_sheet_range.end_label_seq_id
Provider: author / Type: Coordinate replacement
Revision 2.1Oct 2, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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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: Maltose/maltodextrin-binding periplasmic protein,Spizellomyces punctatus Fanzor 1
T: DNA (35-MER)
W: RNA (81-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,1487
Polymers182,0334
Non-polymers1143
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
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: 16652.738 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (35-MER)


Mass: 16616.684 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 Maltose/maltodextrin-binding periplasmic protein,Spizellomyces punctatus Fanzor 1 / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 118029.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria), (gene. exp.) Spizellomyces punctatus (fungus)
Gene: malE, b4034, JW3994, SPPG_07727 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P0AEX9, UniProt: A0A0L0H5U9
#4: RNA chain RNA (81-MER)


Mass: 30734.154 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) / References: GenBank: 1027018667

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Non-polymers , 2 types, 3 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: isolated from a natural source / Formula: Zn

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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: SpuFz1-omegaRNA-target DNA complex / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
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: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 52.51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31906 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 89.82 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00218130
ELECTRON MICROSCOPYf_angle_d0.527511503
ELECTRON MICROSCOPYf_chiral_restr0.04041248
ELECTRON MICROSCOPYf_plane_restr0.00271006
ELECTRON MICROSCOPYf_dihedral_angle_d21.10753618

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