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- PDB-9nvu: Engineered OrufIscB-omegaRNA-target DNA complex -

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Basic information

Entry
Database: PDB / ID: 9nvu
TitleEngineered OrufIscB-omegaRNA-target DNA complex
Components
  • DNA TS
  • NTS
  • OrufIscB-REC-swap 49
  • RNA (162-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / Gene editing / IscB / OMEGA / RNA-guided nuclease / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
metagenome (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsXu, P. / Zhu, S. / Zhang, F.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Biotechnol / Year: 2025
Title: Evolution-guided protein design of IscB for persistent epigenome editing in vivo.
Authors: Soumya Kannan / Han Altae-Tran / Shiyou Zhu / Peiyu Xu / Daniel Strebinger / Rachel Oshiro / Guilhem Faure / Lukas Moeller / Julie Pham / Kepler S Mears / Heyuan M Ni / Rhiannon K Macrae / Feng Zhang /
Abstract: Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is ...Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is difficult to engineer variants with enhanced activity while maintaining specificity. Here we engineer the compact Obligate Mobile Element Guided Activity (OMEGA) RNA-guided endonuclease IscB and its guiding RNA (ωRNA) by combining ortholog screening, structure-guided protein domain design and RNA engineering, and deep learning-based structure prediction to generate an improved variant, NovaIscB. We show that the compact NovaIscB achieves up to 40% indel activity (~100-fold improvement over wild-type OgeuIscB) on the human genome with improved specificity relative to existing IscBs. We further show that NovaIscB can be fused with a methyltransferase to create a programmable transcriptional repressor, OMEGAoff, that is compact enough to be packaged in a single adeno-associated virus vector for persistent in vivo gene repression. This study highlights the power of combining natural diversity with protein engineering to design enhanced enzymes for molecular biology applications.
History
DepositionMar 21, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
N: NTS
P: OrufIscB-REC-swap 49
T: DNA TS
W: RNA (162-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,46310
Polymers163,2354
Non-polymers2286
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA chain , 2 types, 2 molecules NT

#1: DNA chain NTS


Mass: 8885.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#3: DNA chain DNA TS


Mass: 12030.771 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules PW

#2: Protein OrufIscB-REC-swap 49


Mass: 69258.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)
#4: RNA chain RNA (162-MER)


Mass: 73060.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 6 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Engineered OrufIscB-omegaRNA-target DNA complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: metagenome (others)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 48.82 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68817 / Symmetry type: POINT

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