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- PDB-6je4: Crystal structure of Nme1Cas9-sgRNA-dsDNA dimer mediated by doubl... -

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Basic information

Entry
Database: PDB / ID: 6je4
TitleCrystal structure of Nme1Cas9-sgRNA-dsDNA dimer mediated by double protein inhibitor AcrIIC3 monomers
Components
  • AcrIIC3
  • CRISPR-associated endonuclease Cas9
  • non-target DNA strand
  • sgRNA
  • target DNA strand
KeywordsHYDROLASE/HYDROLASE INHIBITOR/DNA/RNA / CRISPR-Cas9 / NmeCas9 / Nme1Cas9 / AcrIIC3 / anti-CRISPR / HYDROLASE / HYDROLASE-HYDROLASE INHIBITOR-DNA-RNA 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 / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DI(HYDROXYETHYL)ETHER / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / : / Uncharacterized protein / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesNeisseria meningitidis 8013 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.069 Å
AuthorsSun, W. / Yang, J. / Cheng, Z. / Liu, C. / Wang, K. / Huang, X. / Wang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China31725008 China
CitationJournal: Mol.Cell / Year: 2019
Title: Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States.
Authors: Sun, W. / Yang, J. / Cheng, Z. / Amrani, N. / Liu, C. / Wang, K. / Ibraheim, R. / Edraki, A. / Huang, X. / Wang, M. / Wang, J. / Liu, L. / Sheng, G. / Yang, Y. / Lou, J. / Sontheimer, E.J. / Wang, Y.
History
DepositionFeb 4, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 8, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Nov 22, 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
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 Cas9
C: target DNA strand
D: non-target DNA strand
B: sgRNA
I: AcrIIC3
E: CRISPR-associated endonuclease Cas9
G: target DNA strand
H: non-target DNA strand
F: sgRNA
J: AcrIIC3
K: CRISPR-associated endonuclease Cas9
M: target DNA strand
N: non-target DNA strand
L: sgRNA
O: CRISPR-associated endonuclease Cas9
Q: target DNA strand
R: non-target DNA strand
P: sgRNA
S: AcrIIC3
T: AcrIIC3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)812,63841
Polymers811,20320
Non-polymers1,43621
Water1,13563
1
A: CRISPR-associated endonuclease Cas9
C: target DNA strand
D: non-target DNA strand
B: sgRNA
J: AcrIIC3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,0318
Polymers202,8015
Non-polymers2303
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24490 Å2
ΔGint-168 kcal/mol
Surface area75200 Å2
MethodPISA
2
I: AcrIIC3
E: CRISPR-associated endonuclease Cas9
G: target DNA strand
H: non-target DNA strand
F: sgRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,21711
Polymers202,8015
Non-polymers4166
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area25230 Å2
ΔGint-161 kcal/mol
Surface area76170 Å2
MethodPISA
3
K: CRISPR-associated endonuclease Cas9
M: target DNA strand
N: non-target DNA strand
L: sgRNA
T: AcrIIC3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,46515
Polymers202,8015
Non-polymers66510
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area26460 Å2
ΔGint-154 kcal/mol
Surface area75850 Å2
MethodPISA
4
O: CRISPR-associated endonuclease Cas9
Q: target DNA strand
R: non-target DNA strand
P: sgRNA
S: AcrIIC3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)202,9257
Polymers202,8015
Non-polymers1242
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24160 Å2
ΔGint-173 kcal/mol
Surface area75300 Å2
MethodPISA
Unit cell
Length a, b, c (Å)129.199, 118.010, 323.986
Angle α, β, γ (deg.)90.00, 89.92, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 2 types, 8 molecules AEKOIJST

#1: Protein
CRISPR-associated endonuclease Cas9


Mass: 124589.883 Da / Num. of mol.: 4 / Mutation: D16A,H588A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis 8013 (bacteria) / Strain: 8013 / Gene: cas9, NMV_1993 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: C9X1G5, Hydrolases; Acting on ester bonds
#5: Protein
AcrIIC3


Mass: 13457.375 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis 8013 (bacteria) / Strain: 8013 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A3E2QDI5, UniProt: A0A425B395*PLUS

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DNA chain , 2 types, 8 molecules CGMQDHNR

#2: DNA chain
target DNA strand


Mass: 10836.049 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain
non-target DNA strand


Mass: 10858.044 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain , 1 types, 4 molecules BFLP

#4: RNA chain
sgRNA


Mass: 43059.352 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 84 molecules

#6: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: C2H6O2
#7: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 63 / 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: 3.04 Å3/Da / Density % sol: 59.6 %
Crystal growTemperature: 289 K / Method: evaporation / pH: 5.8
Details: 1M NaCl, 0.1M citric pH 5.76, 30% PEG 600, 9.6% glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 1.06 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Dec 31, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.06 Å / Relative weight: 1
ReflectionResolution: 3.06→50 Å / Num. obs: 178199 / % possible obs: 98.5 % / Redundancy: 4.3 % / CC1/2: 0.98 / Rmerge(I) obs: 0.133 / Rpim(I) all: 0.069 / Rrim(I) all: 0.15 / Χ2: 0.925 / Net I/σ(I): 9.4
Reflection shellResolution: 3.06→3.11 Å / Redundancy: 3.9 % / Rmerge(I) obs: 0.701 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 8728 / CC1/2: 0.508 / Rpim(I) all: 0.384 / Rrim(I) all: 0.804 / Χ2: 0.802 / % possible all: 97.4

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Processing

Software
NameVersionClassification
PHENIX(1.14_3247: ???)refinement
HKL-3000data reduction
HKL-3000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6JDQ

6jdq


Resolution: 3.069→46.284 Å / SU ML: 0.33 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 24.62
RfactorNum. reflection% reflection
Rfree0.2409 8047 5.03 %
Rwork0.2101 --
obs0.2117 159906 87.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.069→46.284 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms36931 15437 93 63 52524
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00454967
X-RAY DIFFRACTIONf_angle_d0.83777595
X-RAY DIFFRACTIONf_dihedral_angle_d12.73233377
X-RAY DIFFRACTIONf_chiral_restr0.0479028
X-RAY DIFFRACTIONf_plane_restr0.0057391
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.069-3.10390.36911460.31972398X-RAY DIFFRACTION42
3.1039-3.14040.31021760.31023084X-RAY DIFFRACTION54
3.1404-3.17870.31021830.28493287X-RAY DIFFRACTION57
3.1787-3.21890.32372060.2743539X-RAY DIFFRACTION62
3.2189-3.26130.34281870.28413827X-RAY DIFFRACTION66
3.2613-3.30590.29232060.2684021X-RAY DIFFRACTION70
3.3059-3.35320.27472440.26324212X-RAY DIFFRACTION74
3.3532-3.40320.28462340.25244465X-RAY DIFFRACTION78
3.4032-3.45630.34822300.26524772X-RAY DIFFRACTION82
3.4563-3.5130.25252430.2594950X-RAY DIFFRACTION86
3.513-3.57350.29023080.2565115X-RAY DIFFRACTION89
3.5735-3.63850.29952540.25235290X-RAY DIFFRACTION92
3.6385-3.70850.2513160.24135355X-RAY DIFFRACTION94
3.7085-3.78410.25942800.23635566X-RAY DIFFRACTION96
3.7841-3.86640.26323020.23025544X-RAY DIFFRACTION98
3.8664-3.95630.24662790.22315740X-RAY DIFFRACTION98
3.9563-4.05510.24193250.21365697X-RAY DIFFRACTION99
4.0551-4.16470.2373450.20195633X-RAY DIFFRACTION99
4.1647-4.28720.2162960.19245684X-RAY DIFFRACTION99
4.2872-4.42550.22862930.18845701X-RAY DIFFRACTION99
4.4255-4.58350.21382820.18575778X-RAY DIFFRACTION99
4.5835-4.76680.1992660.17585765X-RAY DIFFRACTION99
4.7668-4.98350.2323080.18175744X-RAY DIFFRACTION99
4.9835-5.24590.21272780.18835761X-RAY DIFFRACTION100
5.2459-5.57410.24193130.19425796X-RAY DIFFRACTION100
5.5741-6.00360.23712930.20075792X-RAY DIFFRACTION100
6.0036-6.60630.2433230.20355784X-RAY DIFFRACTION100
6.6063-7.55860.22052780.18665866X-RAY DIFFRACTION100
7.5586-9.50950.19973300.1745803X-RAY DIFFRACTION100
9.5095-46.28890.20853230.19785890X-RAY DIFFRACTION98

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