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- EMDB-40184: Structure of the Spizellomyces punctatus Fanzor (SpuFz) in comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-40184
TitleStructure of the Spizellomyces punctatus Fanzor (SpuFz) in complex with omega RNA and target DNA
Map data
Sample
  • Complex: SpuFz-wRNA-tgDNA complex
    • DNA: DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')
    • Protein or peptide: Maltodextrin-binding protein (Fragment),OrfB_Zn_ribbon domain-containing protein
    • DNA: DNA (35-MER)
    • RNA: RNA (78-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: water
KeywordsFanzor / Eukaryotic / RNA-guided / nuclease / Gene editing / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyMaltose/Cyclodextrin ABC transporter, substrate-binding protein / carbohydrate transmembrane transporter activity / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Maltodextrin-binding protein / Uncharacterized protein
Function and homology information
Biological speciesSpizellomyces punctatus (fungus) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsXu P / Saito M / Zhang F
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40184.png

Downloads & links

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Map

FileDownload / File: emd_40184.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.033429183 - 2.2004864
Average (Standard dev.)0.0011892542 (±0.02550147)
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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Additional map: #1

Fileemd_40184_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_40184_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40184_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SpuFz-wRNA-tgDNA complex

EntireName: SpuFz-wRNA-tgDNA complex
Components
  • Complex: SpuFz-wRNA-tgDNA complex
    • DNA: DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*GP*GP*GP*CP*AP*TP*A)-3')
    • Protein or peptide: Maltodextrin-binding protein (Fragment),OrfB_Zn_ribbon domain-containing protein
    • DNA: DNA (35-MER)
    • RNA: RNA (78-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: SpuFz-wRNA-tgDNA complex

SupramoleculeName: SpuFz-wRNA-tgDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Spizellomyces punctatus (fungus)

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Macromolecule #1: 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: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.594986 KDa
SequenceString:
(DC)(DG)(DG)(DT)(DA)(DC)(DC)(DC)(DG)(DG) (DG)(DC)(DA)(DT)(DA)

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Macromolecule #3: DNA (35-MER)

MacromoleculeName: DNA (35-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.737919 KDa
SequenceString:
(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)

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Macromolecule #2: Maltodextrin-binding protein (Fragment),OrfB_Zn_ribbon domain-con...

MacromoleculeName: Maltodextrin-binding protein (Fragment),OrfB_Zn_ribbon domain-containing protein
type: protein_or_peptide / ID: 2 / 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: Maltodextrin-binding protein, Uncharacterized protein

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Macromolecule #4: RNA (78-MER)

MacromoleculeName: RNA (78-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Spizellomyces punctatus (fungus)
Molecular weightTheoretical: 25.050807 KDa
SequenceString:
UCCGAGCCGG UAGUCGCGCG GUUCAAUCCC UGGUGCGGGU GCUAGUGCAC CGGCUCCGCA CUAUCUAUAG GUUAUGAA

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 3 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 501142
FSC plot (resolution estimation)

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