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- PDB-5vgb: Crystal structure of NmeCas9 HNH domain bound to anti-CRISPR AcrIIC1 -

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Basic information

Entry
Database: PDB / ID: 5vgb
TitleCrystal structure of NmeCas9 HNH domain bound to anti-CRISPR AcrIIC1
Components
  • Anti-CRISPR protein (AcrIIC1)
  • CRISPR-associated endonuclease Cas9
KeywordsHYDROLASE/HYDROLASE inhibitor / Protein / RNA / HYDROLASE-HYDROLASE inhibitor complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
Anti-CRISPR protein (AcrIIC1) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesNeisseria meningitidis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.497 Å
AuthorsHarrington, L.B. / Doxzen, K.W. / Ma, E. / Knott, G.J. / Kranzusch, P.J. / Doudna, J.A.
CitationJournal: Cell / Year: 2017
Title: A Broad-Spectrum Inhibitor of CRISPR-Cas9.
Authors: Harrington, L.B. / Doxzen, K.W. / Ma, E. / Liu, J.J. / Knott, G.J. / Edraki, A. / Garcia, B. / Amrani, N. / Chen, J.S. / Cofsky, J.C. / Kranzusch, P.J. / Sontheimer, E.J. / Davidson, A.R. / ...Authors: Harrington, L.B. / Doxzen, K.W. / Ma, E. / Liu, J.J. / Knott, G.J. / Edraki, A. / Garcia, B. / Amrani, N. / Chen, J.S. / Cofsky, J.C. / Kranzusch, P.J. / Sontheimer, E.J. / Davidson, A.R. / Maxwell, K.L. / Doudna, J.A.
History
DepositionApr 10, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 30, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Sep 20, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 13, 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

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: Anti-CRISPR protein (AcrIIC1)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,8166
Polymers26,4442
Non-polymers3724
Water2,612145
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2780 Å2
ΔGint-16 kcal/mol
Surface area12010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)46.164, 60.487, 77.320
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 16805.801 Da / Num. of mol.: 1 / Fragment: HNH domain (UNP residues 518-655)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis (bacteria) / Gene: cas9, NMA510612_0701 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: X5EPV9, Hydrolases; Acting on ester bonds
#2: Protein Anti-CRISPR protein (AcrIIC1)


Mass: 9637.853 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis (bacteria) / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A2D0TCG3*PLUS
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 145 / 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: 2.05 Å3/Da / Density % sol: 39.94 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, hanging drop / pH: 4.25
Details: 200 mM Ammonium sulfate, 0.1 M Sodium acetate, 22% PEG 4,000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.11583 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 2, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.11583 Å / Relative weight: 1
ReflectionResolution: 1.49→47.64 Å / Num. obs: 35654 / % possible obs: 99.4 % / Redundancy: 6.4 % / Biso Wilson estimate: 16.64 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.103 / Rpim(I) all: 0.044 / Rrim(I) all: 0.113 / Net I/σ(I): 9.8 / Num. measured all: 228832 / Scaling rejects: 42
Reflection shell

Diffraction-ID: 1

Resolution (Å)% possible obs (%)Redundancy (%)Rmerge(I) obsNum. measured obsNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs
1.49-1.52895.52.505837715260.4111.1412.7641
8.18-47.645.90.02816282770.9990.0120.03129.5

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Phasing

PhasingMethod: SAD

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Processing

Software
NameVersionClassificationNB
PHENIX1.10.1_2155refinement
Aimless0.5.31data scaling
SOLVEphasing
PDB_EXTRACT3.22data extraction
XDSdata reduction
RefinementMethod to determine structure: SAD / Resolution: 1.497→47.641 Å / SU ML: 0.18 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 22.13 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2098 1765 5 %
Rwork0.1642 33544 -
obs0.1665 35309 99.25 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 138.02 Å2 / Biso mean: 30.6462 Å2 / Biso min: 12.03 Å2
Refinement stepCycle: final / Resolution: 1.497→47.641 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1842 0 23 145 2010
Biso mean--51.62 37.6 -
Num. residues----225
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0061998
X-RAY DIFFRACTIONf_angle_d0.8162693
X-RAY DIFFRACTIONf_chiral_restr0.104265
X-RAY DIFFRACTIONf_plane_restr0.004358
X-RAY DIFFRACTIONf_dihedral_angle_d20.035789
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 (%)
1.4974-1.53790.34581230.29772344246791
1.5379-1.58320.32021340.252525452679100
1.5832-1.63430.29551330.212725532686100
1.6343-1.69270.24861350.185925662701100
1.6927-1.76040.21241350.156225522687100
1.7604-1.84060.2011350.147925912726100
1.8406-1.93760.19531360.14625732709100
1.9376-2.0590.1891350.14125602695100
2.059-2.2180.20651370.136126042741100
2.218-2.44120.18781370.143426082745100
2.4412-2.79440.17881380.160926152753100
2.7944-3.52050.21141400.159626602800100
3.5205-47.66520.20581470.170627732920100

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