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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 / homologyCRISPR associated protein Cas1 / CRISPR-associated protein (Cas_Cas2CT1978) / CRISPR-associated protein Cas1 / CRISPR-associated protein Cas2 subtype / CRISPR-associated protein Cas1, ECOLI subtype / CRISPR-associated protein Cas1, type I-E / crossover junction endodeoxyribonuclease activity / 5'-flap endonuclease activity / maintenance of CRISPR repeat elements / defense response to virus ...CRISPR associated protein Cas1 / CRISPR-associated protein (Cas_Cas2CT1978) / CRISPR-associated protein Cas1 / CRISPR-associated protein Cas2 subtype / CRISPR-associated protein Cas1, ECOLI subtype / CRISPR-associated protein Cas1, type I-E / crossover junction endodeoxyribonuclease activity / 5'-flap endonuclease activity / maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Acting on Ester Bonds / cellular response to DNA damage stimulus / DNA repair / DNA binding / identical protein binding / metal ion binding / cytoplasm / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated endonuclease Cas1
Function and homology information
Specimen sourceEscherichia coli (E. coli)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / 3.31 Å resolution
AuthorsWright, A.V. / Knott, G.J. / Doxzen, K.D. / 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: May 19, 2017 / Release: Aug 2, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Aug 2, 2017Structure modelrepositoryInitial release
1.1Sep 27, 2017Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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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
Polyers199,92710
Non-polymers1,29122
Water0
1


  • idetical with deposited unit
  • defined by author&software
  • Evidence: homology, PDB 46PI and 5DS45, gel filtration
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)31540
ΔGint (kcal/M)-368
Surface area (Å2)62550
MethodPISA
Unit cell
γ
α
β
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-MP 1 21 1

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Components

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

#1: Protein/peptide
CRISPR-associated endonuclease Cas1


Mass: 33570.770 Da / Num. of mol.: 4
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, Acting on Ester Bonds
#2: Protein/peptide CRISPR-associated endoribonuclease Cas2


Mass: 11553.270 Da / Num. of mol.: 2
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, 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: (synth.) synthetic construct (others)
#4: DNA chain DNA (11-MER)


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


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


Mass: 17967.461 Da / Num. of mol.: 1 / 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 / Formula: Ni / Nickel

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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 / Density percent sol: 63.08 %
Crystal growTemp: 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 kelvins
SourceSource: SYNCHROTRON / Type: SSRL BEAMLINE BL9-2 / Synchrotron site: SSRL / Beamline: BL9-2 / Wavelength: 0.9795
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Collection date: Mar 4, 2017
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionB iso Wilson estimate: 92.52 Å2 / D resolution high: 2.96 Å / D resolution low: 39.55 Å / Number obs: 47810 / CC half: 0.996 / Rmerge I obs: 0.203 / Rpim I all: 0.083 / Rrim I all: 0.22 / NetI over sigmaI: 7.9 / Number measured all: 329343 / Redundancy: 6.9 % / Scaling rejects: 0 / Percent possible obs: 96.3
Reflection shell

Diffraction ID: 1

Rmerge I obsHighest resolutionLowest resolutionCC halfRpim I allRrim I allRedundancyPercent possible all
4.4102.9603.0700.2151.8424.7926.30071.800
0.03711.48039.5500.9990.0160.0406.50095.700

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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
RefineMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5DS5
Overall SU ML: 0.47 / Cross valid method: FREE R-VALUE / Sigma F: 1.33 / Overall phase error: 28.46
Solvent computationSolvent shrinkage radii: 0.9 Å / Solvent vdw probe radii: 1.11 Å
Displacement parametersB iso max: 328.62 Å2 / B iso mean: 106.5272 Å2 / B iso min: 46.84 Å2
Least-squares processR factor R free: 0.2629 / R factor R work: 0.2243 / R factor obs: 0.2262 / Highest resolution: 3.31 Å / Lowest resolution: 39.504 Å / Number reflection R free: 1833 / Number reflection obs: 35241 / Percent reflection R free: 5.2 / Percent reflection obs: 98.83
Refine hist #finalHighest resolution: 3.31 Å / Lowest resolution: 39.504 Å / B iso mean ligand: 131.45 / Number residues total: 1361
Number of atoms included #finalProtein: 9757 / Nucleic acid: 1983 / Ligand: 22 / Solvent: 0 / Total: 11762
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
Refine LS shell

Refine ID: X-RAY DIFFRACTION / R factor R free error: 0 / Total number of bins used: 13

Highest resolutionR factor R freeR factor R workLowest resolutionNumber reflection R freeNumber reflection R workNumber reflection allPercent reflection obs
3.31000.37020.35333.39951602543270399.0000
3.39950.36830.32613.49941622509267199.0000
3.49940.32200.30473.61231372557269499.0000
3.61230.33860.30023.74131342610274499.0000
3.74130.32920.26393.89101352559269499.0000
3.89100.27360.24294.06791452549269499.0000
4.06790.28240.21764.28211292561269099.0000
4.28210.21670.19444.550114725692716100.0000
4.55010.19420.17884.90081402606274699.0000
4.90080.25540.20585.39291362559269599.0000
5.39290.30910.22546.17071482590273899.0000
6.17070.25760.20817.76471452578272399.0000
7.76470.19000.188439.50641152618273398.0000

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