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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 / 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 / CRISPR-associated endonuclease Cas1, N-terminal domain / CRISPR-associated endonuclease Cas1, C-terminal domain / HU Protein; Chain A / IHF-like DNA-binding proteins ...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 / CRISPR-associated endonuclease Cas1, N-terminal domain / CRISPR-associated endonuclease Cas1, C-terminal domain / HU Protein; Chain A / IHF-like DNA-binding proteins / 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 / Ribosomal Protein L15; Chain: K; domain 2 / Ribosomal Protein L15; Chain: K; domain 2 / Four Helix Bundle (Hemerythrin (Met), subunit A) / Few Secondary Structures / Irregular / Up-down Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / Integration host factor subunit alpha / Integration host factor subunit beta / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated endonuclease Cas1
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Escherichia coli S88 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsWright, A.V. / Liu, J.J. / Nogales, E. / Doudna, J.A.
Funding support United States, 2items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 2, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Sep 27, 2017Group: Database references / Experimental preparation / Category: citation / em_sample_support
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_sample_support.grid_type
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
Polymers253,90812
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area45600 Å2
ΔGint-251 kcal/mol
Surface area86400 Å2

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Components

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

#1: Protein
CRISPR-associated endonuclease Cas1


Mass: 33235.418 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
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, Hydrolases; Acting on ester bonds
#2: Protein CRISPR-associated endoribonuclease Cas2


Mass: 11553.270 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: ygbF, cas2, b2754, JW5438 / Production host: Escherichia coli (E. coli)
References: UniProt: P45956, Hydrolases; 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 method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (45-MER)


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


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


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

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

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


Mass: 11373.952 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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 Integration host factor subunit beta / Lambda phage / IHF-beta


Mass: 10671.178 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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 / 3D reconstruction method: single particle reconstruction

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Sample preparation

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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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) / Num. of grids imaged: 1 / Num. 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 selectionNum. of particles selected: 650000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86000 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALmodel building for protein part
2AB INITIO MODELREALmodel building for DNA part
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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