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- EMDB-27533: Structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-27533
TitleStructure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with omega RNA and target DNA
Map data
Sample
  • Complex: DtIsrB-wRNA-tgDNA complex
    • Protein or peptide: Ubiquitin-like protein SMT3,IsrB protein,monomeric superfolder Green Fluorescent Protein
    • RNA: omega RNA
    • DNA: target DNA
    • DNA: non-target DNA
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMOylation of SUMOylation proteins / SUMO is proteolytically processed / SUMOylation of transcription factors / Postmitotic nuclear pore complex (NPC) reformation / SUMOylation of transcription cofactors / septin ring / SUMOylation of DNA damage response and repair proteins ...SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMOylation of SUMOylation proteins / SUMO is proteolytically processed / SUMOylation of transcription factors / Postmitotic nuclear pore complex (NPC) reformation / SUMOylation of transcription cofactors / septin ring / SUMOylation of DNA damage response and repair proteins / SUMOylation of RNA binding proteins / SUMOylation of DNA replication proteins / SUMOylation of chromatin organization proteins / ubiquitin-like protein ligase binding / protein sumoylation / condensed nuclear chromosome / protein tag activity / identical protein binding / nucleus
Similarity search - Function
Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Ubiquitin-like protein SMT3
Similarity search - Component
Biological speciessynthetic construct (others) / Desulfovirgula thermocuniculi (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSeiichi H / Kappel K / Zhang F
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2022
Title: Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA.
Authors: Seiichi Hirano / Kalli Kappel / Han Altae-Tran / Guilhem Faure / Max E Wilkinson / Soumya Kannan / F Esra Demircioglu / Rui Yan / Momoko Shiozaki / Zhiheng Yu / Kira S Makarova / Eugene V ...Authors: Seiichi Hirano / Kalli Kappel / Han Altae-Tran / Guilhem Faure / Max E Wilkinson / Soumya Kannan / F Esra Demircioglu / Rui Yan / Momoko Shiozaki / Zhiheng Yu / Kira S Makarova / Eugene V Koonin / Rhiannon K Macrae / Feng Zhang /
Abstract: RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies. ...RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.
History
DepositionJul 8, 2022-
Header (metadata) releaseOct 19, 2022-
Map releaseOct 19, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27533.png

Downloads & links

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Map

FileDownload / File: emd_27533.map.gz / Format: CCP4 / Size: 20.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.144 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.07450263 - 0.15367469
Average (Standard dev.)0.00023998306 (±0.005556192)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions176176176
Spacing176176176
CellA=B=C: 201.34401 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27533_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27533_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 : DtIsrB-wRNA-tgDNA complex

EntireName: DtIsrB-wRNA-tgDNA complex
Components
  • Complex: DtIsrB-wRNA-tgDNA complex
    • Protein or peptide: Ubiquitin-like protein SMT3,IsrB protein,monomeric superfolder Green Fluorescent Protein
    • RNA: omega RNA
    • DNA: target DNA
    • DNA: non-target DNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: DtIsrB-wRNA-tgDNA complex

SupramoleculeName: DtIsrB-wRNA-tgDNA complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Macromolecule #1: Ubiquitin-like protein SMT3,IsrB protein,monomeric superfolder Gr...

MacromoleculeName: Ubiquitin-like protein SMT3,IsrB protein,monomeric superfolder Green Fluorescent Protein
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 84.080859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KMSDSEVNQE AKPEVKPEVK PETHINLKVS DGSSEIFFK IKKTTPLRRL MEAFAKRQGK EMDSLRFLYD GIRIQADQTP EDLDMEDNDI IEAHREQIGG SMSTSITRVP V VGVDGRPL ...String:
MGSSHHHHHH SSGLVPRGSH MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KMSDSEVNQE AKPEVKPEVK PETHINLKVS DGSSEIFFK IKKTTPLRRL MEAFAKRQGK EMDSLRFLYD GIRIQADQTP EDLDMEDNDI IEAHREQIGG SMSTSITRVP V VGVDGRPL MPTTPRKARL LIRDGLAVPR RNKLGLFYIQ MLRPVGTRTQ PVALAVDPGA KYDGVAVASH RRVELRAMVF LP DDVPRKM ETRRNLRRAR RYRKTPRRPA RFDNRRRKGY WLAPTQRFKV EARLKVVREL CRIYPVQLIV TEDVRFNHAR DRN GKYFST VEIGKTLTYR EYRKLAELRL VEVSETDAWR ERFGLEKRTE RKCEQVPETH ANDAAAMLMG VTGCAHNPAA PFFV WRRLR YARRSLFRQN PQKDGVRPRF GGTANGGFFR KGDWVEAEKA GKVYRGWVCG LPTETTKLVG VADADGKRIG QFSPK KVRL LARSTGFSWK EVAAHSSPEV SKGEELFTGV VPILVELDGD VNGHKFSVRG EGEGDATNGK LTLKFICTTG KLPVPW PTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTISF KDDGTYKTRA EVKFEGDTLV NRIELKGIDF KEDGNIL GH KLEYNFNSHN VYITADKQKN GIKANFKIRH NVEDGSVQLA DHYQQNTPIG DGPVLLPDNH YLSTQSKLSK DPNEKRDH M VLLEFVTAAG ITLGMDELYK

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Macromolecule #2: omega RNA

MacromoleculeName: omega RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Desulfovirgula thermocuniculi (bacteria)
Molecular weightTheoretical: 91.748258 KDa
SequenceString: GGGCCUUAUU AAAUGACUUC UCGUCAACCA CCCCUGACUG AAGUCAGAGG CUUGCUUCUG GCCUGAGUUG GGGGCCCGGU UUGGCGGGG CCGGGGGCAA CUGGCUGACC AGGCGGCCCG GUUCGCCGGG CAGGGGUCCG CGGGGCUACC AAGGACUUCC G GGUGUUUC ...String:
GGGCCUUAUU AAAUGACUUC UCGUCAACCA CCCCUGACUG AAGUCAGAGG CUUGCUUCUG GCCUGAGUUG GGGGCCCGGU UUGGCGGGG CCGGGGGCAA CUGGCUGACC AGGCGGCCCG GUUCGCCGGG CAGGGGUCCG CGGGGCUACC AAGGACUUCC G GGUGUUUC GCCAGCCCGG ACUAUCUCCG GCAGAACCGC UCAAUGCCGC GGCCGGCCAA GACCGGCCUA AGCCCUGCGG AC AGCGCCG AGGCGACAAU CACUCCGAAA GGAGGCCGUG UAUCGGC

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Macromolecule #3: target DNA

MacromoleculeName: target DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.593223 KDa
SequenceString:
(DG)(DA)(DT)(DC)(DA)(DG)(DC)(DT)(DC)(DA) (DA)(DG)(DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA) (DT)(DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG) (DG)(DC)

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Macromolecule #4: non-target DNA

MacromoleculeName: non-target DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.075026 KDa
SequenceString:
(DT)(DT)(DG)(DA)(DG)(DC)(DT)(DG)(DA)(DT)

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 58188
FSC plot (resolution estimation)

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