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- PDB-5u30: Crystal structure of AacC2c1-sgRNA-extended target DNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 5u30
TitleCrystal structure of AacC2c1-sgRNA-extended target DNA ternary complex
Components
  • CRISPR-associated endonuclease C2c1
  • Non-target DNA strand
  • Target DNA strand
  • sgRNA
KeywordsHYDROLASE/DNA / Type V CRISPR-Cas endonculease: C2c1: Structure: Binary complex with sgRNA: Ternary complex with added DNA: RuvC catalytic pocket: Sequence-specific PAM recognition: Genome editing tool / HYDROLASE-DNA complex
Function / homology
Function and homology information


defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding
Similarity search - Function
: / : / : / : / : / : / CRISPR-associated endonuclease C2c1 Nuc-II domain / C2c1 CRISPR-Cas endonuclease, RuvC-like domain / CRISPR-associated endonuclease C2c1 second helical domain / CRISPR-associated endonuclease C2c1 first helical domain / CRISPR-associated endonuclease C2c1, wedge domain, second region
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas12b
Similarity search - Component
Biological speciesAlicyclobacillus acidoterrestris (bacteria)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.92 Å
AuthorsYang, H. / Gao, P. / Rajashankar, K.R. / Patel, D.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM104962 United States
Memorial Sloan-Kettering Cancer Center Core GrantP30 CA008748 United States
CitationJournal: Cell / Year: 2016
Title: PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.
Authors: Yang, H. / Gao, P. / Rajashankar, K.R. / Patel, D.J.
History
DepositionDec 1, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease C2c1
B: sgRNA
C: Target DNA strand
D: Non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,2727
Polymers181,9844
Non-polymers2883
Water1267
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24540 Å2
ΔGint-227 kcal/mol
Surface area65530 Å2
MethodPISA
Unit cell
Length a, b, c (Å)116.747, 184.807, 214.287
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number23
Space group name H-MI222

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain Target DNA strand


Mass: 11604.422 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: DNA chain Non-target DNA strand


Mass: 2448.613 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein CRISPR-associated endonuclease C2c1 / AacC2c1


Mass: 131747.031 Da / Num. of mol.: 1 / Fragment: CRISPR-associated endonuclease AacC2c1 / Mutation: D570A/E848A/D977A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alicyclobacillus acidoterrestris (bacteria)
Strain: ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B
Gene: c2c1, N007_06525 / Plasmid: pRSFDuet-SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: T0D7A2, Hydrolases; Acting on ester bonds
#2: RNA chain sgRNA


Mass: 36183.555 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Alicyclobacillus acidoterrestris (bacteria)

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Non-polymers , 2 types, 10 molecules

#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 7 / 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: 3.19 Å3/Da / Density % sol: 61.39 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.2
Details: 0.2 M ammonium acetate, 0.1 M HEPES (pH 7.5), and 25% PEG3350 (v/v)

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Oct 12, 2016 / Details: DECTRIS PILATUS 6M-F
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.92→50 Å / Num. obs: 50264 / % possible obs: 99.8 % / Redundancy: 5.5 % / Biso Wilson estimate: 81.56 Å2 / CC1/2: 0.998 / Net I/σ(I): 10.1
Reflection shellResolution: 2.92→3.02 Å / Redundancy: 5.6 % / Mean I/σ(I) obs: 0.8 / CC1/2: 0.368 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
XDSdata scaling
PDB_EXTRACT3.22data extraction
XDSdata reduction
PHASERphasing
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.92→49.351 Å / SU ML: 0.47 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.46 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2441 2422 4.82 %
Rwork0.2054 --
obs0.2073 50251 99.3 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.92→49.351 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8864 3021 15 7 11907
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00212449
X-RAY DIFFRACTIONf_angle_d0.44817461
X-RAY DIFFRACTIONf_dihedral_angle_d20.1797106
X-RAY DIFFRACTIONf_chiral_restr0.0341931
X-RAY DIFFRACTIONf_plane_restr0.0031752
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.92-2.97960.38261360.35072718X-RAY DIFFRACTION97
2.9796-3.04440.34531380.3382746X-RAY DIFFRACTION98
3.0444-3.11520.37431380.35752765X-RAY DIFFRACTION99
3.1152-3.19310.44051570.32442791X-RAY DIFFRACTION100
3.1931-3.27940.30511140.28682810X-RAY DIFFRACTION100
3.2794-3.37590.32531570.25472783X-RAY DIFFRACTION100
3.3759-3.48480.28841360.24192848X-RAY DIFFRACTION100
3.4848-3.60930.28911470.22332765X-RAY DIFFRACTION100
3.6093-3.75380.24391400.22042818X-RAY DIFFRACTION99
3.7538-3.92460.26211400.21142802X-RAY DIFFRACTION99
3.9246-4.13140.22281330.18572808X-RAY DIFFRACTION99
4.1314-4.39010.20621470.16222827X-RAY DIFFRACTION100
4.3901-4.72880.19721260.16082852X-RAY DIFFRACTION100
4.7288-5.20420.20561710.17032806X-RAY DIFFRACTION100
5.2042-5.95620.20841360.18222864X-RAY DIFFRACTION99
5.9562-7.49990.22941530.20322857X-RAY DIFFRACTION99
7.4999-49.35790.21851530.17932969X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: -27.7078 Å / Origin y: -25.3264 Å / Origin z: -40.8344 Å
111213212223313233
T0.5948 Å2-0.0723 Å20.0447 Å2-0.488 Å2-0.0497 Å2--0.5176 Å2
L1.0816 °20.1814 °20.3758 °2-0.5788 °20.2773 °2--0.6146 °2
S-0.0118 Å °-0.0066 Å °0.1464 Å °-0.017 Å °-0.0948 Å °0.2309 Å °0.1043 Å °-0.1786 Å °0.0974 Å °
Refinement TLS groupSelection details: all

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