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- EMDB-45522: Spizellomyces punctatus Fanzor (SpuFz) State 4 -

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Basic information

Entry
Database: EMDB / ID: EMD-45522
TitleSpizellomyces punctatus Fanzor (SpuFz) State 4
Map data
Sample
  • Complex: SpuFz-wRNA-tgDNA complex state 6
    • DNA: DNA (5'-D(*(MG)*(MG)P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
    • DNA: DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,Spizellomyces punctatus Fanzor 1
    • DNA: DNA (29-MER)
    • RNA: RNA (76-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsFanzor / 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 / maltose binding / carbohydrate transport / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / 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 / maltose binding / carbohydrate transport / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / 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
Uncharacterized protein / Maltose/maltodextrin-binding periplasmic protein
Similarity search - Component
Biological speciesSpizellomyces punctatus (fungus) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsXu P / Saito M / Zhang F
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45522.png

Downloads & links

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Map

FileDownload / File: emd_45522.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.2 Å		0.83 Å/pix.
x 256 pix.
= 211.2 Å		0.83 Å/pix.
x 256 pix.
= 211.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.0524689 - 1.743506
Average (Standard dev.)0.0025820849 (±0.057652287)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : SpuFz-wRNA-tgDNA complex state 6

EntireName: SpuFz-wRNA-tgDNA complex state 6
Components
  • Complex: SpuFz-wRNA-tgDNA complex state 6
    • DNA: DNA (5'-D(*(MG)*(MG)P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
    • DNA: DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,Spizellomyces punctatus Fanzor 1
    • DNA: DNA (29-MER)
    • RNA: RNA (76-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: SpuFz-wRNA-tgDNA complex state 6

SupramoleculeName: SpuFz-wRNA-tgDNA complex state 6 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Spizellomyces punctatus (fungus)

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Macromolecule #1: DNA (5'-D(*(MG)*(MG)P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A...

MacromoleculeName: DNA (5'-D(*(MG)*(MG)P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.966352 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)

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Macromolecule #2: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.517935 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)

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

MacromoleculeName: DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 16.652738 KDa
SequenceString: (DA)(DT)(DT)(DC)(DG)(DA)(DG)(DC)(DT)(DC) (DG)(DG)(DT)(DA)(DC)(DC)(DC)(DG)(DG)(DG) (DC)(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DT) (DA)(DG)(DG)(DT)(DT)(DA)(DT)(DG)(DA)(DA) (DA) (DT)(DC)(DA)(DA)(DA)(DT) ...String:
(DA)(DT)(DT)(DC)(DG)(DA)(DG)(DC)(DT)(DC) (DG)(DG)(DT)(DA)(DC)(DC)(DC)(DG)(DG)(DG) (DC)(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DT) (DA)(DG)(DG)(DT)(DT)(DA)(DT)(DG)(DA)(DA) (DA) (DT)(DC)(DA)(DA)(DA)(DT)(DT)(DA) (DC)(DA)(DA)(DA)(DT)(DA)

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Macromolecule #5: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 16.616684 KDa
SequenceString: (DT)(DA)(DT)(DT)(DT)(DG)(DT)(DA)(DA)(DT) (DT)(DT)(DG)(DA)(DT)(DT)(DT)(DC)(DA)(DT) (DA)(DA)(DC)(DC)(DT)(DA)(DT)(DA)(DG) (DA)(DT)(DA)(DT)(DG)(DC)(DC)(DC)(DG)(DG) (DG) (DT)(DA)(DC)(DC)(DG)(DA) ...String:
(DT)(DA)(DT)(DT)(DT)(DG)(DT)(DA)(DA)(DT) (DT)(DT)(DG)(DA)(DT)(DT)(DT)(DC)(DA)(DT) (DA)(DA)(DC)(DC)(DT)(DA)(DT)(DA)(DG) (DA)(DT)(DA)(DT)(DG)(DC)(DC)(DC)(DG)(DG) (DG) (DT)(DA)(DC)(DC)(DG)(DA)(DG)(DC) (DT)(DC)(DG)(DA)(DA)(DT)

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Macromolecule #4: Maltose/maltodextrin-binding periplasmic protein,Spizellomyces pu...

MacromoleculeName: Maltose/maltodextrin-binding periplasmic protein,Spizellomyces punctatus Fanzor 1
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Spizellomyces punctatus (fungus)
Molecular weightTheoretical: 118.029922 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MKSSHHHHHH HHHHGSSMKI EEGKLVIWIN GDKGYNGLAE VGKKFEKDTG IKVTVEHPDK LEEKFPQVAA TGDGPDIIFW AHDRFGGYA QSGLLAEITP DKAFQDKLYP FTWDAVRYNG KLIAYPIAVE ALSLIYNKDL LPNPPKTWEE IPALDKELKA K GKSALMFN ...String:
MKSSHHHHHH HHHHGSSMKI EEGKLVIWIN GDKGYNGLAE VGKKFEKDTG IKVTVEHPDK LEEKFPQVAA TGDGPDIIFW AHDRFGGYA QSGLLAEITP DKAFQDKLYP FTWDAVRYNG KLIAYPIAVE ALSLIYNKDL LPNPPKTWEE IPALDKELKA K GKSALMFN LQEPYFTWPL IAADGGYAFK YENGKYDIKD VGVDNAGAKA GLTFLVDLIK NKHMNADTDY SIAEAAFNKG ET AMTINGP WAWSNIDTSK VNYGVTVLPT FKGQPSKPFV GVLSAGINAA SPNKELAKEF LENYLLTDEG LEAVNKDKPL GAV ALKSYE EELAKDPRIA ATMENAQKGE IMPNIPQMSA FWYAVRTAVI NAASGRQTVD EALKDAQTGS ENLYFQSNAP PKKK QKLER LKKLDKPTLH TCNKTSFAKA FLPNETYRQR LLDYIAIIHQ LADHASHALK FYILSTSTSS FPVVHEDTIE AILYL LNKG EAWHPRKEAK KAWRDCLLPY VQRYCQIVGF IHPNLRGEQQ SINYLTVSMM TNLKVNVQEH FMQMLLRYIN LRFDVK GQK QRLPPKSDAR KAFFTRLRYL KSVFLFDVVP ELEFLDDLTP LESEVLEEIW SLDLPFLPND PLAYAIVADP MSFFPAY CK LSGLYEQYGF QRFSAIPLRR SLIQSHVRID TIILYQHILC ITRRDAETVE KDDLWMRVCN LCTKAFRSRC GMHFEGSI T TDGASVSVYL KHPEADKYGK RGARKSANTV AAEVKALYVE NNLPACRAAE NVVVIDPNKR DILYCQDSNG TTFRYTANQ RAVETGSRRF AKRREAMKEE AGVDLIESRI PSHKTMNLMD FTRYLLVRRA DWDRRKEFYS HPAHTRWKWH SFINRQKSES DLISNMRNK YGENFTVVMG DWSDAGRTAR FQTSSKTKGW RTLFKRNRID CFLLDEYKTS SVCPRCSSSE FVEKKFKTRP H SRPWRRRE GKIEKVHGLL GCTNPNCLQQ AWTSGMRYWN RDMLSTCNML LIVRSMLDGH GRPEVFSRSV PAVA

UniProtKB: Maltose/maltodextrin-binding periplasmic protein, Uncharacterized protein

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Macromolecule #6: RNA (76-MER)

MacromoleculeName: RNA (76-MER) / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Spizellomyces punctatus (fungus)
Molecular weightTheoretical: 30.734154 KDa
SequenceString:
GUUUUCCGAG CCGGUUGUCG CGCGGUUCAA UCCCUGGUGC GGGUGCUAGU GCCAAUACCC ACCGGCUCCG CACUAUCUAU AGGUUAUGA AAUCAAA

GENBANK: GENBANK: XM_016755885.1

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 51.41 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 34628
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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