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- PDB-5mga: Structure of the Cpf1 endonuclease R-loop complex after DNA cleavage -

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Basic information

Entry
Database: PDB / ID: 5mga
TitleStructure of the Cpf1 endonuclease R-loop complex after DNA cleavage
Components
  • CRISPR-associated endonuclease Cpf1
  • DNA (26-MER)
  • DNA (5'-D(P*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
  • RNA (40-MER)
KeywordsHYDROLASE / FnCpf1 / Cpf1 / R-loop / CRISPR
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity
Similarity search - Function
: / CRISPR-associated endonuclease Cpf1 PI domain / : / CRISPR-associated endonuclease Cpf1 REC2 domain / CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida U112 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 3 Å
AuthorsMontoya, G. / Stella, S.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
CitationJournal: Nature / Year: 2017
Title: Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.
Authors: Stella, S. / Alcon, P. / Montoya, G.
History
DepositionNov 21, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 21, 2017Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_id_ASTM ..._citation.country / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 24, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_validate_close_contact / struct_conn / struct_conn_type
Revision 1.3May 8, 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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cpf1
B: RNA (40-MER)
C: DNA (26-MER)
D: DNA (5'-D(P*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)179,3316
Polymers179,2824
Non-polymers492
Water2,342130
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16510 Å2
ΔGint-106 kcal/mol
Surface area66230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)85.223, 137.652, 320.513
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain DNA (26-MER)


Mass: 7937.139 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)
#4: DNA chain DNA (5'-D(P*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')


Mass: 3726.453 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein CRISPR-associated endonuclease Cpf1 / FnCpf1


Mass: 154809.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. novicida U112 (bacteria)
Gene: cpf1, FTN_1397 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: A0Q7Q2, Hydrolases; Acting on ester bonds
#2: RNA chain RNA (40-MER)


Mass: 12808.594 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)

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Non-polymers , 2 types, 132 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 130 / 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: 2.62 Å3/Da / Density % sol: 53.08 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: 0.35M sodium thiocyanate and 20.9% w/v PEG 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 15, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.95→80.12 Å / Num. obs: 39708 / % possible obs: 98 % / Redundancy: 6.7 % / Biso Wilson estimate: 65.4 Å2 / Net I/σ(I): 7
Reflection shellResolution: 2.95→2.96 Å / Redundancy: 7 % / Rmerge(I) obs: 1.75 / Mean I/σ(I) obs: 1.3 / CC1/2: 0.35 / % possible all: 98

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Processing

Software
NameVersionClassification
REFMAC5.8.0155refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementResolution: 3→39.58 Å / Cor.coef. Fo:Fc: 0.923 / Cor.coef. Fo:Fc free: 0.888 / SU B: 63.13 / SU ML: 0.504 / Cross valid method: THROUGHOUT / ESU R Free: 0.459 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2653 1843 4.9 %RANDOM
Rwork0.2416 ---
obs0.2428 35724 98.2 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 97.109 Å2
Baniso -1Baniso -2Baniso -3
1-0.09 Å20 Å20 Å2
2--0.32 Å20 Å2
3----0.41 Å2
Refinement stepCycle: 1 / Resolution: 3→39.58 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9596 1624 2 130 11352
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.01811581
X-RAY DIFFRACTIONr_bond_other_d0.0040.0210198
X-RAY DIFFRACTIONr_angle_refined_deg1.6491.82715908
X-RAY DIFFRACTIONr_angle_other_deg1.287323587
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.92651157
X-RAY DIFFRACTIONr_dihedral_angle_2_deg43.8625.562498
X-RAY DIFFRACTIONr_dihedral_angle_3_deg20.499151833
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.5551530
X-RAY DIFFRACTIONr_chiral_restr0.1010.21685
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.0211869
X-RAY DIFFRACTIONr_gen_planes_other0.0030.022635
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.0334.8744701
X-RAY DIFFRACTIONr_mcbond_other2.0334.8734700
X-RAY DIFFRACTIONr_mcangle_it3.6287.2815831
X-RAY DIFFRACTIONr_mcangle_other3.6287.2815832
X-RAY DIFFRACTIONr_scbond_it1.5514.6116880
X-RAY DIFFRACTIONr_scbond_other1.5514.616879
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.846.88910078
X-RAY DIFFRACTIONr_long_range_B_refined9.18984.10247627
X-RAY DIFFRACTIONr_long_range_B_other9.17884.07447598
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 3→3.077 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.396 115 -
Rwork0.397 2572 -
obs--97.57 %
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)
10.8693-0.11620.41290.7372-0.71671.8046-0.1674-0.0787-0.14070.25790.1054-0.0937-0.18480.04090.0620.12160.0646-0.02390.0471-0.01020.926817.91991.2885-43.2034
21.2963-0.93640.93243.2904-0.81561.0119-0.32-0.1577-0.10440.60490.324-0.0537-0.3058-0.1281-0.0040.31490.2050.00560.14210.0190.621912.43958.1248-35.3698
30.4629-0.4139-0.1830.45370.22040.1527-0.1287-0.1946-0.12440.18840.30730.07880.12850.1643-0.17860.22130.2158-0.05910.32330.05421.07312.6901-2.5882-45.2338
47.54821.53010.25760.39130.38312.55630.2744-0.6432-0.77460.1598-0.0905-0.20470.69440.3197-0.18390.28390.1821-0.05290.3530.05460.834220.58715.8571-70.1676
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A3 - 1300
2X-RAY DIFFRACTION2B-19 - 20
3X-RAY DIFFRACTION3C-20 - 5
4X-RAY DIFFRACTION4D-5 - 6

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