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- PDB-5u31: Crystal structure of AacC2c1-sgRNA-8mer substrate DNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 5u31
TitleCrystal structure of AacC2c1-sgRNA-8mer substrate 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 / SAD / Resolution: 2.89 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 1.1Feb 8, 2017Group: Structure summary
Revision 1.2Sep 20, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jan 1, 2020Group: Author supporting evidence / Database references / Category: pdbx_audit_support / struct_ref_seq
Item: _pdbx_audit_support.funding_organization / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end
Revision 1.4Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
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 C2c1
B: sgRNA
C: Target DNA strand
D: Non-target DNA strand
E: Non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)181,5527
Polymers181,3605
Non-polymers1922
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24220 Å2
ΔGint-205 kcal/mol
Surface area65660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)117.249, 182.152, 216.789
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number23
Space group name H-MI222

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Components

#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)
#3: DNA chain Target DNA strand


Mass: 8532.490 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.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
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: 3.2 Å3/Da / Density % sol: 61.52 %
Crystal growTemperature: 293 K / Method: evaporation / 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-E / Wavelength: 0.9792 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Aug 22, 2016 / Details: LR-Design detector positioner
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.89→50 Å / Num. obs: 52212 / % possible obs: 100 % / Redundancy: 14.3 % / Biso Wilson estimate: 59.26 Å2 / CC1/2: 0.996 / Net I/σ(I): 11.7
Reflection shellResolution: 2.89→2.98 Å / Redundancy: 11.8 % / Mean I/σ(I) obs: 1.2 / CC1/2: 0.48 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
XDSdata scaling
PDB_EXTRACT3.22data extraction
XDSdata reduction
AutoSolphasing
SCALEPACKdata scaling
RefinementMethod to determine structure: SAD / Resolution: 2.89→49.295 Å / SU ML: 0.42 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 25.74 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2393 2555 4.89 %
Rwork0.1976 --
obs0.1997 52212 99.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.89→49.295 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8864 2997 10 0 11871
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00412415
X-RAY DIFFRACTIONf_angle_d0.6217413
X-RAY DIFFRACTIONf_dihedral_angle_d20.7597091
X-RAY DIFFRACTIONf_chiral_restr0.041925
X-RAY DIFFRACTIONf_plane_restr0.0041751
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.89-2.94560.37041360.33712715X-RAY DIFFRACTION100
2.9456-3.00570.31971480.30842748X-RAY DIFFRACTION100
3.0057-3.07110.40241160.29732703X-RAY DIFFRACTION100
3.0711-3.14250.32841500.29132751X-RAY DIFFRACTION100
3.1425-3.22110.33661500.28292686X-RAY DIFFRACTION100
3.2211-3.30810.34051340.24372729X-RAY DIFFRACTION100
3.3081-3.40550.27631480.22662751X-RAY DIFFRACTION100
3.4055-3.51540.25261340.21152733X-RAY DIFFRACTION100
3.5154-3.6410.25541490.20232748X-RAY DIFFRACTION100
3.641-3.78670.23481450.18712739X-RAY DIFFRACTION100
3.7867-3.9590.22581330.17682752X-RAY DIFFRACTION100
3.959-4.16760.20031470.1722765X-RAY DIFFRACTION100
4.1676-4.42850.19141340.1562750X-RAY DIFFRACTION100
4.4285-4.77020.19041370.15222773X-RAY DIFFRACTION100
4.7702-5.24980.21171380.16262792X-RAY DIFFRACTION100
5.2498-6.00830.21491300.17262795X-RAY DIFFRACTION100
6.0083-7.56540.2311620.19162802X-RAY DIFFRACTION100
7.5654-49.30220.20631640.18582925X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: 145.0204 Å / Origin y: 207.3176 Å / Origin z: 284.3392 Å
111213212223313233
T0.4264 Å2-0.0295 Å2-0.0082 Å2-0.3447 Å20.0315 Å2--0.3479 Å2
L1.0401 °20.1875 °2-0.3648 °2-0.5221 °2-0.2095 °2--0.5083 °2
S-0.0215 Å °0.0776 Å °-0.1136 Å °-0.0577 Å °-0.0431 Å °-0.1892 Å °-0.0112 Å °0.1114 Å °0.0589 Å °
Refinement TLS groupSelection details: all

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