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

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Basic information

Entry
Database: EMDB / ID: 8827
TitleCas1-Cas2-IHF-DNA holo-complex
Map dataCas1-Cas2-IHF-DNA holo-complex
SampleCas1-Cas2-IHF-DNA holo complex
  • (CRISPR-associated ...) x 2
  • (nucleic-acidNucleic acid) x 4
  • (Integration host factor subunit ...Lambda phage) x 2
Function / homologyHistone-like DNA-binding protein / CRISPR-associated protein Cas2 subtype / Integration host factor, alpha subunit / CRISPR-associated protein Cas1 / Integration host factor (IHF)-like DNA-binding domain superfamily / CRISPR-associated protein Cas1, ECOLI subtype / Histone-like DNA-binding protein, conserved site / CRISPR-associated protein Cas1, type I-E / Bacterial DNA-binding protein / CRISPR associated protein Cas1 ...Histone-like DNA-binding protein / CRISPR-associated protein Cas2 subtype / Integration host factor, alpha subunit / CRISPR-associated protein Cas1 / Integration host factor (IHF)-like DNA-binding domain superfamily / CRISPR-associated protein Cas1, ECOLI subtype / Histone-like DNA-binding protein, conserved site / CRISPR-associated protein Cas1, type I-E / Bacterial DNA-binding protein / CRISPR associated protein Cas1 / CRISPR-associated protein (Cas_Cas2CT1978) / Bacterial histone-like DNA-binding proteins signature. / Integration host factor, beta subunit / crossover junction endodeoxyribonuclease activity / 5'-flap endonuclease activity / maintenance of CRISPR repeat elements / defense response to virus / chromosome / regulation of translation / endonuclease activity / DNA recombination / Acting on Ester Bonds / cellular response to DNA damage stimulus / DNA repair / regulation of transcription, DNA-templated / transcription, DNA-templated / DNA binding / identical protein binding / metal ion binding / cytoplasm / Integration host factor subunit alpha / Integration host factor subunit beta / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated endonuclease Cas1
Function and homology information
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 3.64 Å resolution
AuthorsWright AV / Liu JJ
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.
Validation ReportPDB-ID: 5wfe

SummaryFull reportAbout validation report
DateDeposition: Jul 11, 2017 / Header (metadata) release: Aug 2, 2017 / Map release: Aug 2, 2017 / Last update: Sep 27, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
  • Download
  • 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

Downloads & links

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Map

Fileemd_8827.map.gz (map file in CCP4 format, 108001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
300 pix
1.07 Å/pix.
= 321. Å
300 pix
1.07 Å/pix.
= 321. Å
300 pix
1.07 Å/pix.
= 321. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour Level:0.8 (by author), 0.8 (movie #1):
Minimum - Maximum-2.0546746 - 4.251078
Average (Standard dev.)0.00078146334 (0.08141421)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions300300300
Origin-150-150-150
Limit149149149
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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 / Number of components: 9

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Component #1: protein, Cas1-Cas2-IHF-DNA holo complex

ProteinName: Cas1-Cas2-IHF-DNA holo complex / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, CRISPR-associated endonuclease Cas1

ProteinName: CRISPR-associated endonuclease Cas1 / Details: Cas1 / Recombinant expression: No
MassTheoretical: 33.235418 kDa
Source (engineered)Expression System: Escherichia coli K-12 (bacteria) / Strain: K12

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Component #3: protein, CRISPR-associated endoribonuclease Cas2

ProteinName: CRISPR-associated endoribonuclease Cas2 / Recombinant expression: No
MassTheoretical: 11.55327 kDa
Source (engineered)Expression System: Escherichia coli K-12 (bacteria) / Strain: K12

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Component #4: nucleic-acid, DNA (28-MER)

Nucleic-acidName: DNA (28-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(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)
MassTheoretical: 8.680646 kDa

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Component #5: nucleic-acid, DNA (45-MER)

Nucleic-acidName: DNA (45-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(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)
MassTheoretical: 18.745965 kDa

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Component #6: nucleic-acid, DNA (76-MER)

Nucleic-acidName: DNA (76-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(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)
MassTheoretical: 29.101656 kDa

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Component #7: nucleic-acid, DNA (61-MER)

Nucleic-acidName: DNA (61-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(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)
MassTheoretical: 19.286447 kDa

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Component #8: protein, Integration host factor subunit alpha

ProteinName: Integration host factor subunit alphaLambda phage / Recombinant expression: No
MassTheoretical: 11.373952 kDa
Source (engineered)Expression System: Escherichia coli S88 (bacteria) / Strain: S88 / ExPEC

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Component #9: protein, Integration host factor subunit beta

ProteinName: Integration host factor subunit betaLambda phage / Recombinant expression: No
MassTheoretical: 10.671178 kDa
Source (engineered)Expression System: Escherichia coli S88 (bacteria) / Strain: S88 / ExPEC

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.3 mg/ml
Buffer solution: 20 mM HEPES, pH 7.5, 150 mM KCl, 5 mM EDTA, 1 mM DTT, and 0.1% glycerol
pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 281.15 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.5 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.6 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3000

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 86000
3D reconstructionSoftware: cryoSPARC / Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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