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

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Basic information

Entry
Database: PDB / ID: 5wfe
TitleCas1-Cas2-IHF-DNA holo-complex
Components
  • (CRISPR-associated ...) x 2
  • (Integration host factor subunit ...Lambda phage) x 2
  • DNA (28-MER)
  • DNA (45-MER)
  • DNA (61-MER)
  • DNA (76-MER)
KeywordsDNA BINDING PROTEIN/DNA / CRISPR integration complex / DNA / Cas1-Cas2 / IHF / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
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
Specimen sourceEscherichia coli K-12 (bacteria)
Escherichia coli S88 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.64 Å resolution
AuthorsWright, A.V. / Liu, J.J. / Nogales, E. / Doudna, J.A.
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 Report
SummaryFull reportAbout validation report
DateDeposition: Jul 11, 2017 / Release: Aug 2, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Aug 2, 2017Structure modelrepositoryInitial release
1.1Sep 13, 2017Structure modelAuthor supporting evidence / Data collectionem_software / pdbx_audit_support_em_software.name / _pdbx_audit_support.funding_organization
1.2Sep 27, 2017Structure modelDatabase references / Experimental preparationcitation / em_sample_support_citation.journal_volume / _citation.page_first / _citation.page_last / _em_sample_support.grid_type
1.3Jul 18, 2018Structure modelData collectionem_software_em_software.image_processing_id / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas1
B: CRISPR-associated endonuclease Cas1
C: CRISPR-associated endonuclease Cas1
D: CRISPR-associated endonuclease Cas1
E: CRISPR-associated endoribonuclease Cas2
F: CRISPR-associated endoribonuclease Cas2
G: DNA (28-MER)
H: DNA (45-MER)
I: DNA (76-MER)
J: DNA (61-MER)
K: Integration host factor subunit alpha
L: Integration host factor subunit beta


Theoretical massNumber of molelcules
Total (without water)253,90812
Polyers253,90812
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)45600
ΔGint (kcal/M)-251
Surface area (Å2)86400

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Components

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CRISPR-associated ... , 2 types, 6 molecules ABCDEF

#1: Protein/peptide
CRISPR-associated endonuclease Cas1


Mass: 33235.418 Da / Num. of mol.: 4 / Details: Cas1 / Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: ygbT, cas1, b2755, JW2725 / Production host: Escherichia coli (E. coli) / References: UniProt: Q46896, Acting on Ester Bonds
#2: Protein/peptide CRISPR-associated endoribonuclease Cas2


Mass: 11553.270 Da / Num. of mol.: 2 / Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: ygbF, cas2, b2754, JW5438 / Production host: Escherichia coli (E. coli) / References: UniProt: P45956, Acting on Ester Bonds

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DNA chain , 4 types, 4 molecules GHIJ

#3: DNA chain DNA (28-MER)


Mass: 8680.646 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (45-MER)


Mass: 18745.965 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA (76-MER)


Mass: 29101.656 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (61-MER)


Mass: 19286.447 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct (others)

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Integration host factor subunit ... , 2 types, 2 molecules KL

#7: Protein/peptide Integration host factor subunit alpha / Lambda phage / IHF-alpha


Mass: 11373.952 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli S88 (bacteria) / Strain: S88 / ExPEC / Gene: ihfA, himA, ECS88_1763 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MAS3
#8: Protein/peptide Integration host factor subunit beta / Lambda phage / IHF-beta


Mass: 10671.178 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli S88 (bacteria) / Strain: S88 / ExPEC / Gene: ihfB, himD, ECS88_0940 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MHM1

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Cas1-Cas2-IHF-DNA holo complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionDetails: 20 mM HEPES, pH 7.5, 150 mM KCl, 5 mM EDTA, 1 mM DTT, and 0.1% glycerol
pH: 7.5
SpecimenConc.: 0.3 mg/ml / Details: 1uM Cas1-Cas2-DNA-IHF complexes / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.6 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 1.5 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 3000
Image scansMovie frames/image: 30 / Used frames/image: 3-30

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategoryFitting ID
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting1
10Relion2.1final Euler assignment
11Relion2.1classification
12cryoSPARC3D reconstruction
20PHENIXmodel refinement1
35Cootmodel fitting2
40PHENIXmodel refinement2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 650000
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 86000 / Symmetry type: POINT
Atomic model building
IDDetailsRef protocolRef space
1model building for protein partRIGID BODY FITREAL
2model building for DNA partAB INITIO MODELREAL
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416254
ELECTRON MICROSCOPYf_angle_d0.63722914
ELECTRON MICROSCOPYf_dihedral_angle_d19.9479013
ELECTRON MICROSCOPYf_chiral_restr0.0392602
ELECTRON MICROSCOPYf_plane_restr0.0192305

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