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- EMDB-26782: IscB and wRNA bound to Target DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-26782
TitleIscB and wRNA bound to Target DNA
Map data
Sample
  • Complex: Cryo-EM structure of IscB in complex with RNA and target DNA
    • Protein or peptide: IscBInternational Society for Computational Biology
    • RNA: RNA (222-MER)
    • DNA: DNA target strand
    • DNA: DNA non-target strand
KeywordsCRISPR / IscB / HEARO RNA / omega RNA / RNA BINDING PROTEIN-RNA-DNA complex
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsSchuler GA / Hu C / Ke A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118174 United States
CitationJournal: Science / Year: 2022
Title: Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9.
Authors: Gabriel Schuler / Chunyi Hu / Ailong Ke /
Abstract: Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ...Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.
History
DepositionApr 27, 2022-
Header (metadata) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26782.png

Downloads & links

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Map

FileDownload / File: emd_26782.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.135
Minimum - Maximum-0.390436 - 1.2604398
Average (Standard dev.)-0.0007101762 (±0.025721544)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 299.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #3

Fileemd_26782_additional_1.map
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Additional map: #2

Fileemd_26782_additional_2.map
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Additional map: #1

Fileemd_26782_additional_3.map
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Half map: #2

Fileemd_26782_half_map_1.map
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Half map: #1

Fileemd_26782_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of IscB in complex with RNA and target DNA

EntireName: Cryo-EM structure of IscB in complex with RNA and target DNA
Components
  • Complex: Cryo-EM structure of IscB in complex with RNA and target DNA
    • Protein or peptide: IscBInternational Society for Computational Biology
    • RNA: RNA (222-MER)
    • DNA: DNA target strand
    • DNA: DNA non-target strand

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Supramolecule #1: Cryo-EM structure of IscB in complex with RNA and target DNA

SupramoleculeName: Cryo-EM structure of IscB in complex with RNA and target DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 190 kDa/nm

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Macromolecule #1: IscB

MacromoleculeName: IscB / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 56.688477 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MAVVYVISKS GKPLMPTTRC GHVRILLKEG KARVVERKPF TIQLTYESAE ETQPLVLGID PGRTNIGMSV VTESGESVFN AQIETRNKD VPKLMKDRKQ YRMAHRRLKR RCKRRRRAKA AGTAFEEGEK QRLLPGCFKP ITCKSIRNKE ARFNNRKRPV G WLTPTANH ...String:
MAVVYVISKS GKPLMPTTRC GHVRILLKEG KARVVERKPF TIQLTYESAE ETQPLVLGID PGRTNIGMSV VTESGESVFN AQIETRNKD VPKLMKDRKQ YRMAHRRLKR RCKRRRRAKA AGTAFEEGEK QRLLPGCFKP ITCKSIRNKE ARFNNRKRPV G WLTPTANH LLVTHLNVVK KVQKILPVAK VVLELNRFSF MAMNNPKVQR WQYQRGPLYG KGSVEEAVSM QQDGHCLFCK HG IDHYHHV VPRRKNGSET LENRVGLCEE HHRLVHTDKE WEANLASKKS GMNKKYHALS VLNQIIPYLA DQLADMFPGN FCV TSGQDT YLFREEHGIP KDHYLDAYCI ACSALTDAKK VSSPKGRPYM VHQFRRHDRQ ACHKANLNRS YYMGGKLVAT NRHK AMDQK TDSLEEYRAA HSAADVSKLT VKHPSAQYKD MSRIMPGSIL VSGEGKLFTL SRSEGRNKGQ VNYFVSTEGI KYWAR KCQY LRNNGGLQIY V

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Macromolecule #2: RNA (222-MER)

MacromoleculeName: RNA (222-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 71.877797 KDa
SequenceString: AAAAGAGUGA ACGAGAGGCU CUUCCAACUU UAUGGUUGCG ACCGUAGGUU GAAAGAGCAC AGGCUGAGAC AUUCGUAAGG CCGAAAGAC CGGACGCACC CUGGGAUUUC CCCAGUCCCC GGAACUGCAU AGCGGAUGCC AGUUGAUGGA GCAAUCUAUC A GAUAAGCC ...String:
AAAAGAGUGA ACGAGAGGCU CUUCCAACUU UAUGGUUGCG ACCGUAGGUU GAAAGAGCAC AGGCUGAGAC AUUCGUAAGG CCGAAAGAC CGGACGCACC CUGGGAUUUC CCCAGUCCCC GGAACUGCAU AGCGGAUGCC AGUUGAUGGA GCAAUCUAUC A GAUAAGCC AGGGGGAACA AUCACCUCUC UGUAUCAGAG AGAGUUUUAC AAAAGGAGGA ACGG

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Macromolecule #3: DNA target strand

MacromoleculeName: DNA target strand / type: dna / ID: 3
Details: phosphorothioate (PS) bonds * GCCACGGGCTGACCTCGACTTCTAGT*C*T*C*G*T*T*CACTCTTTTGCCGTACCCTCGTGGGGCG
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.305646 KDa
SequenceString: (DG)(DC)(DC)(DA)(DC)(DG)(DG)(DG)(DC)(DT) (DG)(DA)(DC)(DC)(DT)(DC)(DG)(DA)(DC)(DT) (DT)(DC)(DT)(DA)(DG)(DT)(DC)(DT)(DC) (DG)(DT)(DT)(DC)(DA)(DC)(DT)(DC)(DT)(DT) (DT) (DT)(DG)(DC)(DC)(DG)(DT) ...String:
(DG)(DC)(DC)(DA)(DC)(DG)(DG)(DG)(DC)(DT) (DG)(DA)(DC)(DC)(DT)(DC)(DG)(DA)(DC)(DT) (DT)(DC)(DT)(DA)(DG)(DT)(DC)(DT)(DC) (DG)(DT)(DT)(DC)(DA)(DC)(DT)(DC)(DT)(DT) (DT) (DT)(DG)(DC)(DC)(DG)(DT)(DA)(DC) (DC)(DC)(DT)(DC)(DG)(DT)(DG)(DG)(DG)(DG) (DC)(DC)

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Macromolecule #4: DNA non-target strand

MacromoleculeName: DNA non-target strand / type: dna / ID: 4
Details: phosphorothioate (PS) bond * CGCCCCACGAGGGTACGGCAAAAGA*G*T*T*T*T*T*TTTACTAGAAGTCGAGGTCAGCCCGTGGC
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.557873 KDa
SequenceString: (DC)(DG)(DC)(DC)(DC)(DC)(DA)(DC)(DG)(DA) (DG)(DG)(DG)(DT)(DA)(DC)(DG)(DG)(DC)(DA) (DA)(DA)(DA)(DG)(DA)(DG)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DG) (DA) (DA)(DG)(DT)(DC)(DG)(DA) ...String:
(DC)(DG)(DC)(DC)(DC)(DC)(DA)(DC)(DG)(DA) (DG)(DG)(DG)(DT)(DA)(DC)(DG)(DG)(DC)(DA) (DA)(DA)(DA)(DG)(DA)(DG)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DG) (DA) (DA)(DG)(DT)(DC)(DG)(DA)(DG)(DG) (DT)(DC)(DA)(DG)(DC)(DC)(DC)(DG)(DT)(DG) (DG)(DC)

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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

Concentration0.5 mg/mL
BufferpH: 7.25
Component:
ConcentrationFormulaName
50.0 mMNaClSodium chloridesodium chloride
50.0 mMC8H18N2O4SHEPES
5.0 mMC9H15O6PMagnesium chloride
2.0 mMHOCH2CH2SH2-Mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 100 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 6.5 seconds before plunging.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 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: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 159201

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