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- PDB-4qdl: Crystal structure of E.coli Cas1-Cas2 complex -

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Basic information

Entry
Database: PDB / ID: 4qdl
TitleCrystal structure of E.coli Cas1-Cas2 complex
Components
  • CRISPR-associated endonuclease Cas1
  • CRISPR-associated endoribonuclease Cas2
KeywordsHYDROLASE / CRISPR-Cas / Cas1-Cas2 complex / CRISPR adaptation
Function / homology
Function and homology information


CRISPR-cas system / crossover junction DNA endonuclease activity / 5'-flap endonuclease activity / maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / 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 / endonuclease activity / defense response to virus / 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 Cas1 ...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
CRISPR-associated endoribonuclease Cas2 / CRISPR-associated endonuclease Cas1
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.7 Å
AuthorsTamulaitiene, G. / Sinkunas, T. / Silanskas, A. / Gasiunas, G. / Grazulis, S. / Siksnys, V.
CitationJournal: To be Published
Title: Crystal structure of E.coli Cas1-Cas2 complex
Authors: Tamulaitiene, G. / Sinkunas, T. / Silanskas, A. / Gasiunas, G. / Grazulis, S. / Siksnys, V.
History
DepositionMay 14, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 28, 2014Provider: repository / Type: Initial release
Revision 1.1Mar 7, 2018Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.2Sep 20, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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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


Theoretical massNumber of molelcules
Total (without water)156,4276
Polymers156,4276
Non-polymers00
Water1,00956
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13690 Å2
ΔGint-94 kcal/mol
Surface area49620 Å2
MethodPISA
Unit cell
Length a, b, c (Å)92.764, 127.402, 96.640
Angle α, β, γ (deg.)90.000, 100.890, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
CRISPR-associated endonuclease Cas1


Mass: 33235.418 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K-12 / Gene: YGBT, CAS1, B2755, JW2725 / Plasmid: pCDF / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-AI
References: UniProt: Q46896, Hydrolases; Acting on ester bonds
#2: Protein CRISPR-associated endoribonuclease Cas2


Mass: 11742.494 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K-12 / Gene: YGBF, CAS2, B2754, JW5438 / Plasmid: pCDF / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-AI
References: UniProt: P45956, Hydrolases; Acting on ester bonds
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 56 / Source method: isolated from a natural source / Formula: H2O

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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: 3.58 Å3/Da / Density % sol: 65.69 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: Crystallization buffer was 0.1M Tris-HCl, pH 8.5, 3% (w/v) PEG8000, 6% (v/v) ethylene glycol , VAPOR DIFFUSION, SITTING DROP, temperature 291K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: MAX II / Beamline: I911-3 / Wavelength: 1.001 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Apr 12, 2014 / Details: mirrors
RadiationMonochromator: Si (111) double crystal monochromator, first crystal indirectly water-cooled
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.001 Å / Relative weight: 1
ReflectionResolution: 2.7→47.987 Å / Num. all: 232284 / Num. obs: 60631 / % possible obs: 100 % / Observed criterion σ(F): 1 / Observed criterion σ(I): 1 / Redundancy: 3.8 % / Biso Wilson estimate: 56.29 Å2 / Rsym value: 0.067 / Net I/σ(I): 11.5
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRsym value% possible all
2.7-2.853.80.6951.13386188250.695100
2.85-3.023.80.4041.83200183500.404100
3.02-3.233.80.2632.83011178370.263100
3.23-3.493.80.1435.22805073010.143100
3.49-3.823.80.089.42592267380.08100
3.82-4.273.80.04715.72347661050.047100
4.27-4.933.80.032222063453770.032100
4.93-6.043.80.032221754745820.032100
6.04-8.543.80.02328.71346435420.023100
8.54-47.9873.70.01724721819740.01799.5

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation3.04 Å47.99 Å
Translation3.04 Å47.99 Å

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Processing

Software
NameVersionClassificationNB
SCALA3.3.20data scaling
MOLREP6phasing
PHENIX1.8.3_1479refinement
PDB_EXTRACT3.14data extraction
MxCuBEdata collection
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: pdb entry 3NKD, 4MAK

3nkd

4mak


Resolution: 2.7→47.449 Å / FOM work R set: 0.7534 / SU ML: 0.43 / σ(F): 0.93 / Phase error: 30.97 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.261 5949 9.83 %RANDOM
Rwork0.2268 ---
obs0.2302 118069 98.95 %-
Solvent computationShrinkage radii: 1.1 Å / VDW probe radii: 1.3 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 160.31 Å2 / Biso mean: 64.6 Å2 / Biso min: 27.22 Å2
Refinement stepCycle: LAST / Resolution: 2.7→47.449 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9068 0 0 56 9124
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0079247
X-RAY DIFFRACTIONf_angle_d0.99612596
X-RAY DIFFRACTIONf_chiral_restr0.0391513
X-RAY DIFFRACTIONf_plane_restr0.0071604
X-RAY DIFFRACTIONf_dihedral_angle_d13.4063275
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.7-2.73070.40794050.39723473387898
2.7307-2.76280.40373790.37133565394498
2.7628-2.79650.40783700.37973538390899
2.7965-2.83190.37183740.35883524389898
2.8319-2.86910.38254070.3683530393799
2.8691-2.90840.41514070.36433522392998
2.9084-2.950.35493380.34713520385899
2.95-2.9940.37234240.32313531395599
2.994-3.04080.39383630.313555391898
3.0408-3.09060.32673980.29093554395299
3.0906-3.14390.35453920.2913484387699
3.1439-3.20110.31063940.29513569396399
3.2011-3.26260.31753760.27253538391499
3.2626-3.32920.30113620.26753602396499
3.3292-3.40160.31323520.28953540389299
3.4016-3.48070.31134290.26633511394099
3.4807-3.56770.30254360.23473538397499
3.5677-3.66410.27773860.23523523390999
3.6641-3.77190.26483920.2243507389999
3.7719-3.89360.26163880.21543608399699
3.8936-4.03270.23583870.20493574396199
4.0327-4.19410.18163410.17913601394299
4.1941-4.38480.18923990.17253555395499
4.3848-4.61580.20113930.17453588398199
4.6158-4.90470.20943410.172935843925100
4.9047-5.2830.19463750.171835583933100
5.283-5.81380.21623920.190135843976100
5.8138-6.65310.25164020.212835923994100
6.6531-8.37470.22773930.178235793972100
8.3747-47.45670.20634080.17883519392799

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