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- EMDB-26723: Structure of IsrB ternary complex with RNA mutant and target DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-26723
TitleStructure of IsrB ternary complex with RNA mutant and target DNA
Map data
Sample
  • Complex: IsrB-RNA-DNA complex
    • Protein or peptide: IsrB protein-lambdaN
    • RNA: omegaRNA-boxB
    • DNA: target-DNA
    • DNA: non-target-DNA
Biological speciesDesulfovirgula thermocuniculi (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 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
DepositionApr 22, 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_26723.png

Downloads & links

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Map

FileDownload / File: emd_26723.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.26 Å/pix.
x 160 pix.
= 200.8 Å		1.26 Å/pix.
x 160 pix.
= 200.8 Å		1.26 Å/pix.
x 160 pix.
= 200.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.255 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.73
Minimum - Maximum-0.25337994 - 1.7427977
Average (Standard dev.)0.033618342 (±0.14755921)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 200.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_26723_half_map_1.map
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Half map: #2

Fileemd_26723_half_map_2.map
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Sample components

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Entire : IsrB-RNA-DNA complex

EntireName: IsrB-RNA-DNA complex
Components
  • Complex: IsrB-RNA-DNA complex
    • Protein or peptide: IsrB protein-lambdaN
    • RNA: omegaRNA-boxB
    • DNA: target-DNA
    • DNA: non-target-DNA

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Supramolecule #1: IsrB-RNA-DNA complex

SupramoleculeName: IsrB-RNA-DNA complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Desulfovirgula thermocuniculi (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: IsrB protein-lambdaN

MacromoleculeName: IsrB protein-lambdaN / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfovirgula thermocuniculi (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KMSDSEVNQE AKPEVKPEVK PETHINLKVS DGSSEIFFKI KKTTPLRRLM EAFAKRQGKE MDSLRFLYDG IRIQADQTPE DLDMEDNDII EAHREQIGGS MDAQTRRRER RAEKQAQWKA ...String:
MGSSHHHHHH SSGLVPRGSH MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KMSDSEVNQE AKPEVKPEVK PETHINLKVS DGSSEIFFKI KKTTPLRRLM EAFAKRQGKE MDSLRFLYDG IRIQADQTPE DLDMEDNDII EAHREQIGGS MDAQTRRRER RAEKQAQWKA ANMSTSITRV PVVGVDGRPL MPTTPRKARL LIRDGLAVPR RNKLGLFYIQ MLRPVGTRTQ PVALAVDPGA KYDGVAVASH RRVELRAMVF LPDDVPRKME TRRNLRRARR YRKTPRRPAR FDNRRRKGYW LAPTQRFKVE ARLKVVRELC RIYPVQLIVT EDVRFNHARD RNGKYFSTVE IGKTLTYREY RKLAELRLVE VSETDAWRER FGLEKRTERK CEQVPETHAN DAAAMLMGVT GCAHNPAAPF FVWRRLRYAR RSLFRQNPQK DGVRPRFGGT ANGGFFRKGD WVEAEKAGKV YRGWVCGLPT ETTKLVGVAD ADGKRIGQFS PKKVRLLARS TGFSWKEVAA HSSPEVSKGE ELFTGVVPIL VELDGDVNGH KFSVRGEGEG DATNGKLTLK FICTTGKLPV PWPTLVTTLT YGVQCFSRYP DHMKQHDFFK SAMPEGYVQE RTISFKDDGT YKTRAEVKFE GDTLVNRIEL KGIDFKEDGN ILGHKLEYNF NSHNVYITAD KQKNGIKANF KIRHNVEDGS VQLADHYQQN TPIGDGPVLL PDNHYLSTQS KLSKDPNEKR DHMVLLEFVT AAGITLGMDE LYK

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Macromolecule #2: omegaRNA-boxB

MacromoleculeName: omegaRNA-boxB / type: rna / ID: 2
Source (natural)Organism: Desulfovirgula thermocuniculi (bacteria)
SequenceString: ggGCCUUAUU AAAUGACUUC UCGUCAACCA CCCCUGAAAG GGGGCCCGGU UUGGCGGGGC CGGGGGCAAC UGGCUGACCA GGCGGCCCGG UUCGCCGGGC AGGGGUCCGC GGGGCUACCA AGGACUUCCG GGUGUUUCGC CAGCCCGGAC UAUCUCCGGC AGAACCGCUC ...String:
ggGCCUUAUU AAAUGACUUC UCGUCAACCA CCCCUGAAAG GGGGCCCGGU UUGGCGGGGC CGGGGGCAAC UGGCUGACCA GGCGGCCCGG UUCGCCGGGC AGGGGUCCGC GGGGCUACCA AGGACUUCCG GGUGUUUCGC CAGCCCGGAC UAUCUCCGGC AGAACCGCUC AAUGCCGCGG CCGGCCAAGA CCGGCCUAAG CCCUGCGGAC AGCGCCGAGG CGACAAUCAC UCCGAAAGGA GGCCGUGUAU CGGCGAAAGC CCUGAAGAAG GGC

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Macromolecule #3: target-DNA

MacromoleculeName: target-DNA / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
gatCAGCTCA AGAGAAGTCA TTTAATAAGG C

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Macromolecule #4: non-target-DNA

MacromoleculeName: non-target-DNA / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
TACTGAAGAG TTGAGCTGat

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 0.0026000000000000003 µm / Nominal defocus min: 0.001 µm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 3.1265 e/Å2
Experimental equipment
Model: Talos Arctica / 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: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 50661

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