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- PDB-4r5q: Crystal structure and nuclease activity of the CRISPR-associated ... -

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Basic information

Entry
Database: PDB / ID: 4r5q
TitleCrystal structure and nuclease activity of the CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis containing a [2Fe-2S] cluster
ComponentsCRISPR-associated exonuclease, Cas4 family
KeywordsHYDROLASE / MCSG / STRUCTURAL GENOMICS / PSI-BIOLOGY / EXONUCLEASE / Midwest Center for Structural Genomics
Function / homology
Function and homology information


5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / single-stranded DNA 5'-3' DNA exonuclease activity / single-stranded DNA endodeoxyribonuclease activity / DNA duplex unwinding / 2 iron, 2 sulfur cluster binding / defense response to virus / metal ion binding
Similarity search - Function
: / CRISPR-associated protein Cas4 / Lambda Exonuclease; Chain A - #10 / Dna2/Cas4, domain of unknown function DUF83 / Domain of unknown function DUF83 / Lambda Exonuclease; Chain A / PD-(D/E)XK endonuclease-like domain superfamily / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
FE2/S2 (INORGANIC) CLUSTER / CRISPR-associated exonuclease Cas4
Similarity search - Component
Biological speciesPyrobaculum calidifontis JCM 11548 (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.65 Å
AuthorsNocek, B. / Skarina, T. / Lemak, S. / Brown, G. / Savchenko, A. / Joachimiak, A. / Yakunin, A. / Midwest Center for Structural Genomics (MCSG)
CitationJournal: To be Published
Title: Crystal structure and nuclease activity of the CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis containing a [2Fe-2S] cluster
Authors: Nocek, B. / Skarina, T. / Lemak, S. / Brown, G. / Savchenko, A. / Joachimiak, A. / Yakunin, A.
History
DepositionAug 21, 2014Deposition site: RCSB / Processing site: RCSB
SupersessionSep 17, 2014ID: 4ONB
Revision 1.0Sep 17, 2014Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2017Group: Author supporting evidence / Refinement description / Category: pdbx_struct_assembly_auth_evidence / software
Revision 1.2Nov 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
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_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_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_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_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_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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated exonuclease, Cas4 family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,2313
Polymers25,0311
Non-polymers2002
Water37821
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)117.836, 117.836, 89.412
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number97
Space group name H-MI422
Components on special symmetry positions
IDModelComponents
11A-403-

HOH

DetailsSolution and crystallographic studies indicate monomer as most likely oligomeric state. SEC experiment shows that this protein is a monomer in the solution.

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Components

#1: Protein CRISPR-associated exonuclease, Cas4 family


Mass: 25031.348 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrobaculum calidifontis JCM 11548 (archaea)
Strain: JCM 11548 / VA1 / Gene: Pcal_0546 / Production host: Escherichia coli (E. coli) / Strain (production host): Bl21DE3 / References: UniProt: A3MTK6
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe2S2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 21 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 3.1 Å3/Da / Density % sol: 60.32 %
Crystal growTemperature: 293 K / pH: 7
Details: 3.5M Sodium FORMATE, 0.1M BIS-TRIS PROPANE, pH 7, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.9794
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: May 12, 2012 / Details: MIRRORS
RadiationMonochromator: DOUBLE CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9794 Å / Relative weight: 1
ReflectionResolution: 2.65→37.26 Å / Num. obs: 9378 / % possible obs: 99.4 % / Observed criterion σ(I): 2 / Redundancy: 10.4 % / Biso Wilson estimate: 33 Å2 / Rmerge(I) obs: 0.047 / Net I/σ(I): 41

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Processing

Software
NameVersionClassification
SBC-Collectdata collection
HKL-3000phasing
DMmodel building
SHELXmodel building
Cootmodel building
PHENIX(phenix.refine: 1.8.4_1496)refinement
HKL-3000data reduction
HKL-3000data scaling
DMphasing
SHELXphasing
RefinementMethod to determine structure: SAD / Resolution: 2.65→37.26 Å / SU ML: 0.34 / σ(F): 3 / Phase error: 26.36 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.253 651 4.76 %
Rwork0.228 --
obs0.229 9155 99.1 %
all-9378 -
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.65→37.26 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1711 0 5 21 1737
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.011759
X-RAY DIFFRACTIONf_angle_d0.6322368
X-RAY DIFFRACTIONf_dihedral_angle_d14.45659
X-RAY DIFFRACTIONf_chiral_restr0.028269
X-RAY DIFFRACTIONf_plane_restr0.003303
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.65-2.85570.3864830.36741576X-RAY DIFFRACTION47
2.8557-3.14290.35681200.30822012X-RAY DIFFRACTION61
3.1429-3.59740.29921380.24422893X-RAY DIFFRACTION88
3.5974-4.53110.20781560.19883298X-RAY DIFFRACTION99
4.5311-37.26670.20771540.19943237X-RAY DIFFRACTION97
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
14.4564-0.95330.08632.47750.29372.23080.0784-0.13830.34260.21270.0803-0.0503-0.5243-0.2667-0.11360.64770.2040.27590.4840.04240.4974-1.7209-20.7067-4.6934
25.3842-2.8275-1.21445.22773.56677.10950.3248-0.21980.3871-0.0125-0.1496-0.4311-0.38520.6662-0.09990.3107-0.08840.15690.4561-0.15330.460420.9589-28.4109-10.0368
31.1931-0.0894-0.66240.3086-0.09671.02580.16690.21960.1237-0.0828-0.05930.0032-0.2771-0.37270.05880.28150.02470.27380.341-0.13410.360113.7338-29.8806-25.1499
44.1744-0.5866-1.70740.54930.70391.89010.0766-0.07030.07130.01390.033-0.0304-0.11680.0334-0.03070.29810.10740.17520.3314-0.02430.25774.6646-25.8007-8.5641
53.90190.3184-1.2231.7067-1.18724.00910.11260.6028-0.3327-0.2795-0.01820.00260.0264-0.73470.18390.31890.25380.09390.7734-0.03830.342-5.7286-30.2308-19.0081
63.9245-1.0238-2.02590.71680.76891.17650.29321.25490.0088-0.678-0.2121-0.0713-0.6563-0.8076-0.06620.58570.19120.17660.73720.01120.47764.8215-25.3087-28.7588
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid -2 through 23 )
2X-RAY DIFFRACTION2chain 'A' and (resid 24 through 42 )
3X-RAY DIFFRACTION3chain 'A' and (resid 43 through 83 )
4X-RAY DIFFRACTION4chain 'A' and (resid 84 through 101 )
5X-RAY DIFFRACTION5chain 'A' and (resid 102 through 157 )
6X-RAY DIFFRACTION6chain 'A' and (resid 158 through 212 )

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