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- EMDB-8827: Cas1-Cas2-IHF-DNA holo-complex -

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Basic information

Entry
Database: EMDB / ID: EMD-8827
TitleCas1-Cas2-IHF-DNA holo-complex
Map dataCas1-Cas2-IHF-DNA holo-complex
Sample
  • Complex: Cas1-Cas2-IHF-DNA holo complex
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • DNA: DNA (28-MER)
    • DNA: DNA (45-MER)
    • DNA: DNA (76-MER)
    • DNA: DNA (61-MER)
    • Protein or peptide: Integration host factor subunit alphaLambda phage
    • Protein or peptide: Integration host factor subunit betaLambda phage
KeywordsCRISPR integration complex / DNA / Cas1-Cas2 / IHF / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


CRISPR-cas system / crossover junction DNA endonuclease activity / 5'-flap endonuclease activity / maintenance of CRISPR repeat elements / structural constituent of chromatin / regulation of translation / chromosome / defense response to virus / endonuclease activity / DNA recombination ...CRISPR-cas system / crossover junction DNA endonuclease activity / 5'-flap endonuclease activity / maintenance of CRISPR repeat elements / structural constituent of chromatin / regulation of translation / chromosome / defense response to virus / endonuclease activity / DNA recombination / Hydrolases; Acting on ester bonds / DNA repair / DNA damage response / regulation of DNA-templated transcription / protein homodimerization activity / DNA binding / identical protein binding / metal ion binding / cytoplasm
Similarity search - Function
Integration host factor, alpha subunit / Integration host factor, beta subunit / CRISPR-associated protein Cas2 subtype / CRISPR-associated protein (Cas_Cas2CT1978) / CRISPR-associated protein Cas1, ECOLI subtype / CRISPR-associated protein Cas1, type I-E / Histone-like DNA-binding protein, conserved site / Bacterial histone-like DNA-binding proteins signature. / Histone-like DNA-binding protein / Bacterial DNA-binding protein ...Integration host factor, alpha subunit / Integration host factor, beta subunit / CRISPR-associated protein Cas2 subtype / CRISPR-associated protein (Cas_Cas2CT1978) / CRISPR-associated protein Cas1, ECOLI subtype / CRISPR-associated protein Cas1, type I-E / Histone-like DNA-binding protein, conserved site / Bacterial histone-like DNA-binding proteins signature. / Histone-like DNA-binding protein / Bacterial DNA-binding protein / bacterial (prokaryotic) histone like domain / CRISPR-associated endonuclease Cas1, N-terminal domain / Integration host factor (IHF)-like DNA-binding domain superfamily / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1
Similarity search - Domain/homology
Integration host factor subunit alpha / Integration host factor subunit beta / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated endonuclease Cas1
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli K-12 (bacteria) / Escherichia coli S88 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsWright AV / Liu JJ
Funding support United States, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1244557 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1P50GM102706-01 United States
CitationJournal: Science / Year: 2017
Title: Structures of the CRISPR genome integration complex.
Authors: Addison V Wright / Jun-Jie Liu / Gavin J Knott / Kevin W Doxzen / Eva Nogales / Jennifer A Doudna /
Abstract: CRISPR-Cas systems depend on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses. We present crystal structures of ...CRISPR-Cas systems depend on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses. We present crystal structures of Cas1-Cas2 bound to both donor and target DNA in intermediate and product integration complexes, as well as a cryo-electron microscopy structure of the full CRISPR locus integration complex, including the accessory protein IHF (integration host factor). The structures show unexpectedly that indirect sequence recognition dictates integration site selection by favoring deformation of the repeat and the flanking sequences. IHF binding bends the DNA sharply, bringing an upstream recognition motif into contact with Cas1 to increase both the specificity and efficiency of integration. These results explain how the Cas1-Cas2 CRISPR integrase recognizes a sequence-dependent DNA structure to ensure site-selective CRISPR array expansion during the initial step of bacterial adaptive immunity.
History
DepositionJul 11, 2017-
Header (metadata) releaseAug 2, 2017-
Map releaseAug 2, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5wfe
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8827.png

Downloads & links

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Map

FileDownload / File: emd_8827.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas1-Cas2-IHF-DNA holo-complex
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-2.0546746 - 4.251078
Average (Standard dev.)0.00078146334 (±0.08141421)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z321.000321.000321.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-2.0554.2510.001

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Supplemental data

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Sample components

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Entire : Cas1-Cas2-IHF-DNA holo complex

EntireName: Cas1-Cas2-IHF-DNA holo complex
Components
  • Complex: Cas1-Cas2-IHF-DNA holo complex
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • DNA: DNA (28-MER)
    • DNA: DNA (45-MER)
    • DNA: DNA (76-MER)
    • DNA: DNA (61-MER)
    • Protein or peptide: Integration host factor subunit alphaLambda phage
    • Protein or peptide: Integration host factor subunit betaLambda phage

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Supramolecule #1: Cas1-Cas2-IHF-DNA holo complex

SupramoleculeName: Cas1-Cas2-IHF-DNA holo complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: CRISPR-associated endonuclease Cas1

MacromoleculeName: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 1 / Details: Cas1 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 33.235418 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTWLPLNPIP LKDRVSMIFL QYGQIDVIDG AFVLIDKTGI RTHIPVGSVA CIMLEPGTRV SHAAVRLAAQ VGTLLVWVGE AGVRVYASG QPGGARSDKL LYQAKLALDE DLRLKVVRKM FELRFGEPAP ARRSVEQLRG IEGSRVRATY ALLAKQYGVT W NGRRYDPK ...String:
MTWLPLNPIP LKDRVSMIFL QYGQIDVIDG AFVLIDKTGI RTHIPVGSVA CIMLEPGTRV SHAAVRLAAQ VGTLLVWVGE AGVRVYASG QPGGARSDKL LYQAKLALDE DLRLKVVRKM FELRFGEPAP ARRSVEQLRG IEGSRVRATY ALLAKQYGVT W NGRRYDPK DWEKGDTINQ CISAATSCLY GVTEAAILAA GYAPAIGFVH TGKPLSFVYD IADIIKFDTV VPKAFEIARR NP GEPDREV RLACRDIFRS SKTLAKLIPL IEDVLAAGEI QPPAPPEDAQ PVAIPLPVSL GDAGHRSS

UniProtKB: CRISPR-associated endonuclease Cas1

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Macromolecule #2: CRISPR-associated endoribonuclease Cas2

MacromoleculeName: CRISPR-associated endoribonuclease Cas2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 11.55327 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSMLVVVTEN VPPRLRGRLA IWLLEVRAGV YVGDVSAKIR EMIWEQIAGL AEEGNVVMAW ATNTETGFEF QTFGLNRRTP VDLDGLRLV SFLPVGSSEN LYFQ

UniProtKB: CRISPR-associated endoribonuclease Cas2

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Macromolecule #7: Integration host factor subunit alpha

MacromoleculeName: Integration host factor subunit alpha / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli S88 (bacteria) / Strain: S88 / ExPEC
Molecular weightTheoretical: 11.373952 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MALTKAEMSE YLFDKLGLSK RDAKELVELF FEEIRRALEN GEQVKLSGFG NFDLRDKNQR PGRNPKTGED IPITARRVVT FRPGQKLKS RVENASPKDE

UniProtKB: Integration host factor subunit alpha

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Macromolecule #8: Integration host factor subunit beta

MacromoleculeName: Integration host factor subunit beta / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli S88 (bacteria) / Strain: S88 / ExPEC
Molecular weightTheoretical: 10.671178 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTKSELIERL ATQQSHIPAK TVEDAVKEML EHMASTLAQG ERIEIRGFGS FSLHYRAPRT GRNPKTGDKV ELEGKYVPHF KPGKELRDR ANIYG

UniProtKB: Integration host factor subunit beta

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Macromolecule #3: DNA (28-MER)

MacromoleculeName: DNA (28-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.680646 KDa
SequenceString:
(DA)(DA)(DA)(DC)(DA)(DC)(DC)(DA)(DG)(DA) (DA)(DC)(DG)(DA)(DG)(DT)(DA)(DG)(DT)(DA) (DA)(DA)(DT)(DT)(DG)(DG)(DG)(DC)

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Macromolecule #4: DNA (45-MER)

MacromoleculeName: DNA (45-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.745965 KDa
SequenceString: (DA)(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DC)(DT) (DC)(DG)(DT)(DT)(DC)(DT)(DG)(DG)(DT)(DG) (DT)(DT)(DT)(DC)(DT)(DC)(DG)(DT)(DG) (DT)(DG)(DT)(DT)(DC)(DC)(DC)(DC)(DG)(DC) (DG) (DC)(DC)(DA)(DG)(DC)(DG) ...String:
(DA)(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DC)(DT) (DC)(DG)(DT)(DT)(DC)(DT)(DG)(DG)(DT)(DG) (DT)(DT)(DT)(DC)(DT)(DC)(DG)(DT)(DG) (DT)(DG)(DT)(DT)(DC)(DC)(DC)(DC)(DG)(DC) (DG) (DC)(DC)(DA)(DG)(DC)(DG)(DG)(DG) (DG)(DA)(DT)(DA)(DA)(DA)(DC)(DC)(DG)(DA) (DG)(DC) (DA)

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Macromolecule #5: DNA (76-MER)

MacromoleculeName: DNA (76-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 29.101656 KDa
SequenceString: (DT)(DG)(DC)(DT)(DC)(DG)(DG)(DT)(DT)(DT) (DA)(DT)(DC)(DC)(DC)(DC)(DG)(DC)(DT)(DG) (DG)(DC)(DG)(DC)(DG)(DG)(DG)(DG)(DA) (DA)(DC)(DA)(DC)(DT)(DC)(DT)(DA)(DA)(DA) (DC) (DA)(DT)(DA)(DA)(DC)(DC) ...String:
(DT)(DG)(DC)(DT)(DC)(DG)(DG)(DT)(DT)(DT) (DA)(DT)(DC)(DC)(DC)(DC)(DG)(DC)(DT)(DG) (DG)(DC)(DG)(DC)(DG)(DG)(DG)(DG)(DA) (DA)(DC)(DA)(DC)(DT)(DC)(DT)(DA)(DA)(DA) (DC) (DA)(DT)(DA)(DA)(DC)(DC)(DT)(DA) (DT)(DT)(DA)(DT)(DT)(DA)(DA)(DT)(DT)(DA) (DA)(DT) (DG)(DA)(DT)(DT)(DT)(DT)(DT) (DT)(DA)(DA)(DG)(DC)(DC)(DA)(DG)(DT)(DC) (DA)(DC)(DA) (DA)(DT)(DC)(DT)(DA)(DC) (DC)(DA)(DA)(DC)(DT)(DT)(DT)(DA)(DT)

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Macromolecule #6: DNA (61-MER)

MacromoleculeName: DNA (61-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 19.286447 KDa
SequenceString: (DA)(DT)(DA)(DA)(DA)(DG)(DT)(DT)(DG)(DG) (DT)(DA)(DG)(DA)(DT)(DT)(DG)(DT)(DG)(DA) (DC)(DT)(DG)(DG)(DC)(DT)(DT)(DA)(DA) (DA)(DA)(DA)(DA)(DT)(DC)(DA)(DT)(DT)(DA) (DA) (DT)(DT)(DA)(DA)(DT)(DA) ...String:
(DA)(DT)(DA)(DA)(DA)(DG)(DT)(DT)(DG)(DG) (DT)(DA)(DG)(DA)(DT)(DT)(DG)(DT)(DG)(DA) (DC)(DT)(DG)(DG)(DC)(DT)(DT)(DA)(DA) (DA)(DA)(DA)(DA)(DT)(DC)(DA)(DT)(DT)(DA) (DA) (DT)(DT)(DA)(DA)(DT)(DA)(DA)(DT) (DA)(DG)(DG)(DT)(DT)(DA)(DT)(DG)(DT)(DT) (DT)(DA) (DG)(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

Concentration0.3 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES, pH 7.5, 150 mM KCl, 5 mM EDTA, 1 mM DTT, and 0.1% glycerol
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV
Details1uM Cas1-Cas2-DNA-IHF complexes

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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: 2.6 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 3-30 / Number grids imaged: 1 / Number real images: 3000 / Average exposure time: 6.0 sec. / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 650000
Startup modelType of model: OTHER / Details: Common line
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationSoftware - Name: Relion2.1
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: Relion2.1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 86000

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Atomic model buiding 1

Detailsmodel building for protein part
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5wfe:
Cas1-Cas2-IHF-DNA holo-complex

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Atomic model buiding 2

Detailsmodel building for DNA part
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-5wfe:
Cas1-Cas2-IHF-DNA holo-complex

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