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- PDB-5vvl: Cas1-Cas2 bound to full-site mimic with Ni -

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Basic information

Entry
Database: PDB / ID: 5vvl
TitleCas1-Cas2 bound to full-site mimic with Ni
Components
  • (CRISPR-associated ...) x 2
  • (DNA (11-MER)) x 2
  • (DNA (58-MER)) x 2
KeywordsHYDROLASE/DNA / Complex / DNA / HYDROLASE-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 / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / DNA repair / DNA damage response / protein homodimerization activity ...CRISPR-cas system / crossover junction DNA endonuclease activity / 5'-flap endonuclease activity / maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / DNA repair / DNA damage response / protein homodimerization activity / DNA binding / identical protein binding / metal ion binding / cytoplasm
Similarity search - Function
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 / CRISPR-associated endonuclease Cas1, N-terminal domain / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain ...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 / CRISPR-associated endonuclease Cas1, N-terminal domain / : / 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) / Up-down Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
NICKEL (II) ION / DNA / DNA (> 10) / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated endonuclease Cas1
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.31 Å
AuthorsWright, A.V. / Knott, G.J. / Doxzen, K.D. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1244557 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
DepositionMay 19, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 2, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 4, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_symmetry / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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 (11-MER)
H: DNA (11-MER)
J: DNA (58-MER)
K: DNA (58-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,21832
Polymers199,92710
Non-polymers1,29122
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: homology, PDB 46PI and 5DS45, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31540 Å2
ΔGint-368 kcal/mol
Surface area62550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)74.642, 197.728, 88.792
Angle α, β, γ (deg.)90.000, 111.350, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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

#1: Protein
CRISPR-associated endonuclease Cas1


Mass: 33570.770 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: ygbT, cas1, b2755, JW2725 / Variant: MG1655 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
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 (strain K12) (bacteria)
Strain: K12 / Gene: ygbF, cas2, b2754, JW5438 / Variant: MG1655 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: P45956, Hydrolases; Acting on ester bonds

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DNA chain , 4 types, 4 molecules GHJK

#3: DNA chain DNA (11-MER)


Mass: 3319.175 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (11-MER)


Mass: 3343.200 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA (58-MER)


Mass: 17907.432 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (58-MER)


Mass: 17967.461 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 22 molecules

#7: Chemical...
ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 22
Source method: isolated from a genetically manipulated source
Formula: Ni

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.33 Å3/Da / Density % sol: 63.08 %
Crystal growTemperature: 281 K / Method: vapor diffusion, hanging drop / pH: 6.4
Details: 100 mM MES pH 6.4, 20% (w/v) PEG MME 2000, 0.2 M NaCl, 3 mM NiCl2

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Data collection

DiffractionMean temperature: 80 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 4, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.96→39.55 Å / Num. obs: 47810 / % possible obs: 96.3 % / Redundancy: 6.9 % / Biso Wilson estimate: 92.52 Å2 / CC1/2: 0.996 / Rmerge(I) obs: 0.203 / Rpim(I) all: 0.083 / Rrim(I) all: 0.22 / Net I/σ(I): 7.9 / Num. measured all: 329343 / Scaling rejects: 0
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsCC1/2Rpim(I) allRrim(I) all% possible all
2.96-3.076.34.410.2151.8424.79271.8
11.48-39.556.50.0370.9990.0160.0495.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.11.1_2575refinement
Aimless0.5.31data scaling
PDB_EXTRACT3.22data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5DS5

5ds5


Resolution: 3.31→39.504 Å / SU ML: 0.47 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 28.46
RfactorNum. reflection% reflection
Rfree0.2629 1833 5.2 %
Rwork0.2243 --
obs0.2262 35241 98.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 328.62 Å2 / Biso mean: 106.5272 Å2 / Biso min: 46.84 Å2
Refinement stepCycle: final / Resolution: 3.31→39.504 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9757 1983 22 0 11762
Biso mean--131.45 --
Num. residues----1361
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00312166
X-RAY DIFFRACTIONf_angle_d0.49316903
X-RAY DIFFRACTIONf_chiral_restr0.0371932
X-RAY DIFFRACTIONf_plane_restr0.0031832
X-RAY DIFFRACTIONf_dihedral_angle_d16.6966941
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 13

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.31-3.39950.37021600.35332543270399
3.3995-3.49940.36831620.32612509267199
3.4994-3.61230.3221370.30472557269499
3.6123-3.74130.33861340.30022610274499
3.7413-3.8910.32921350.26392559269499
3.891-4.06790.27361450.24292549269499
4.0679-4.28210.28241290.21762561269099
4.2821-4.55010.21671470.194425692716100
4.5501-4.90080.19421400.17882606274699
4.9008-5.39290.25541360.20582559269599
5.3929-6.17070.30911480.22542590273899
6.1707-7.76470.25761450.20812578272399
7.7647-39.50640.191150.18842618273398

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