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- EMDB-45518: Guillardia theta Fanzor (GtFz) State 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-45518
TitleGuillardia theta Fanzor (GtFz) State 3
Map data
Sample
  • Complex: Guillardia theta Fanzor1-omegaRNA-target DNA complex
    • DNA: DNA (28-MER)
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,Guillardia theta Fanzor1
    • DNA: DNA (5'-D(P*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*TP*AP*AP*AP*GP*GP*CP*CP*CP*CP*GP*GP*G)-3')
    • RNA: RNA (142-MER)
  • Ligand: ZINC 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 / 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 / DNA binding / 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 speciesGuillardia theta (eukaryote) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 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 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45518.png

Downloads & links

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Map

FileDownload / File: emd_45518.map.gz / Format: CCP4 / Size: 103 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 300 pix.
= 247.5 Å		0.83 Å/pix.
x 300 pix.
= 247.5 Å		0.83 Å/pix.
x 300 pix.
= 247.5 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: 0.1
Minimum - Maximum-0.0017960961 - 1.9209038
Average (Standard dev.)0.0012871324 (±0.02521293)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Guillardia theta Fanzor1-omegaRNA-target DNA complex

EntireName: Guillardia theta Fanzor1-omegaRNA-target DNA complex
Components
  • Complex: Guillardia theta Fanzor1-omegaRNA-target DNA complex
    • DNA: DNA (28-MER)
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,Guillardia theta Fanzor1
    • DNA: DNA (5'-D(P*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*TP*AP*AP*AP*GP*GP*CP*CP*CP*CP*GP*GP*G)-3')
    • RNA: RNA (142-MER)
  • Ligand: ZINC ION

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Supramolecule #1: Guillardia theta Fanzor1-omegaRNA-target DNA complex

SupramoleculeName: Guillardia theta Fanzor1-omegaRNA-target DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Guillardia theta (eukaryote)

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Macromolecule #1: DNA (28-MER)

MacromoleculeName: DNA (28-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 24.883936 KDa
SequenceString: (DG)(DA)(DC)(DC)(DA)(DT)(DG)(DA)(DT)(DT) (DA)(DC)(DG)(DC)(DC)(DA)(DA)(DG)(DC)(DT) (DT)(DT)(DT)(DT)(DA)(DA)(DG)(DA)(DG) (DT)(DG)(DG)(DC)(DC)(DT)(DT)(DA)(DT)(DT) (DA) (DA)(DA)(DT)(DG)(DA)(DC) ...String:
(DG)(DA)(DC)(DC)(DA)(DT)(DG)(DA)(DT)(DT) (DA)(DC)(DG)(DC)(DC)(DA)(DA)(DG)(DC)(DT) (DT)(DT)(DT)(DT)(DA)(DA)(DG)(DA)(DG) (DT)(DG)(DG)(DC)(DC)(DT)(DT)(DA)(DT)(DT) (DA) (DA)(DA)(DT)(DG)(DA)(DC)(DT)(DT) (DC)(DT)(DC)(DC)(DT)(DT)(DA)(DA)(DA)(DT) (DG)(DC) (DC)(DC)(DG)(DG)(DG)(DT)(DA) (DC)(DC)(DG)(DA)(DG)(DC)(DT)(DC)(DG)(DA) (DA)(DT)(DT) (DC)

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

MacromoleculeName: DNA (5'-D(P*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*TP*AP*AP*AP*GP*GP*CP*CP*CP*CP*GP*GP*G)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.07107 KDa
SequenceString: (DG)(DA)(DA)(DT)(DT)(DC)(DG)(DA)(DG)(DC) (DT)(DC)(DG)(DG)(DT)(DA)(DC)(DC)(DC)(DG) (DG)(DG)(DC)(DA)(DT)(DT)(DT)(DA)(DA) (DG)(DG)(DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA) (DT) (DT)(DT)(DA)(DA)(DT)(DA) ...String:
(DG)(DA)(DA)(DT)(DT)(DC)(DG)(DA)(DG)(DC) (DT)(DC)(DG)(DG)(DT)(DA)(DC)(DC)(DC)(DG) (DG)(DG)(DC)(DA)(DT)(DT)(DT)(DA)(DA) (DG)(DG)(DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA) (DT) (DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG) (DG)(DC)(DC)(DA)(DC)(DT)(DC)(DT)(DT)(DA) (DA)(DA) (DA)(DA)(DG)(DC)(DT)(DT)(DG) (DG)(DC)(DG)(DT)(DA)(DA)(DT)(DC)(DA)(DT) (DG)(DG)(DT) (DC)

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Macromolecule #2: Maltose/maltodextrin-binding periplasmic protein,Guillardia theta...

MacromoleculeName: Maltose/maltodextrin-binding periplasmic protein,Guillardia theta Fanzor1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Guillardia theta (eukaryote)
Molecular weightTheoretical: 124.818938 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 EALKDAQTNS SSNNNNNNNN NNLG IEENL YFQSNASNIR IVKRKAKGFF KCEDLVTIKD AVKAAHRIMS DASILVRSYY LRWFQSSYPL DSDDKELELE HFHIS MACS IVQGITRPPV RGVGPEQSVK IDVFNDMLDE YKRLYERAPN DKENETDLSL SHVLAYSIDN LLTAYKNNIE AHFSKY VKR FIRCDMLAKG FNKSEANRVA AIYTNAYIYD SSLDLEPDFM ERLGLEATSY SSLFPSKINK GGFPRVYDLK ANPWVYL PK MVMINQALET DFSSVEHKER RLLNPLPFYS SFVPMHIRID TSGLSQLLMT KDRLDDFKRS YLAEFGVSLN IKNKGDML A SFEKIFGRKA TSNREAGLYA TEMWSFLTNL KTCRQWKELD GVVRKNDPKG TQWMFDNAVV TDGVSISFQV IDNSMFGRK AFSGRKKRVA CQEANDEEDS KQVTREELKT SKLLGCDPGK RDILAITDGI KTICYTKGQR DMDTHKTIRL RTSLKRRRGC GLEEYETQV MNRFQKRSCH PEMFRRYACS RKRMEHMLLE CYSHPVFREF KFLVYNKTKS SEHRFMHRVL ETFKRPQTNL S KARCASGV MRMNALKEVQ RHGDIIIGWG NWGKNPNALR CSAGPTPGIG IRRRFESLFK TTTVPEHYTS QECPSCKGRC LR KATGNPI MRHHLLRCTN DSCCSRWWNR NVAGAFNILT RLLDGQTLSG NETTGDGLGG DDL

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

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Macromolecule #4: RNA (142-MER)

MacromoleculeName: RNA (142-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Guillardia theta (eukaryote)
Molecular weightTheoretical: 49.715664 KDa
SequenceString:
CACUAUCCGG UAACGAAACU ACCGGAGACG GGUUAGGAGG UGACGACCUC UAAAACCUAG AACUUAGAGU GCAAAAACGC CAUUACGAU UGUGAUGCCU AUUCAAGGGU GUCCCAAGUG UAAAAAGAAA GCACUCUAAG GAGUGGCCUU AUUAA

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 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: 58.18 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: 2.5 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: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 328609
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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