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- PDB-5u33: Crystal structure of AacC2c1-sgRNA-extended non-target DNA ternar... -

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Basic information

Entry
Database: PDB / ID: 5u33
TitleCrystal structure of AacC2c1-sgRNA-extended non-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: 3.75 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 / Database references / Category: pdbx_audit_support / struct_ref_seq
Item: _pdbx_audit_support.funding_organization / _struct_ref_seq.db_align_end
Revision 1.3Oct 4, 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

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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)184,1075
Polymers184,0114
Non-polymers961
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23240 Å2
ΔGint-195 kcal/mol
Surface area65980 Å2
MethodPISA
Unit cell
Length a, b, c (Å)113.495, 179.108, 216.706
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: 130762.211 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 (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) (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: 8532.469 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
Compound detailsthe position of TTTT could not be definitely determined within the poly-T sequence of the non- ...the position of TTTT could not be definitely determined within the poly-T sequence of the non-target DNA strand. The poly T fragment is thus arbitrary numbered 101-118.

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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: 3.06 Å3/Da / Density % sol: 59.86 %
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-C / Wavelength: 0.9791 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: 0.9791 Å / Relative weight: 1
ReflectionResolution: 3.75→50 Å / Num. obs: 21992 / % possible obs: 99.8 % / Redundancy: 5.9 % / Biso Wilson estimate: 134.01 Å2 / CC1/2: 0.995 / Net I/σ(I): 5.2
Reflection shellResolution: 3.75→4.05 Å / Redundancy: 5.7 % / Mean I/σ(I) obs: 0.6 / CC1/2: 0.399 / % possible all: 99.7

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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
Starting model: 5U31

5u31


Resolution: 3.75→47.492 Å / SU ML: 0.58 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 31.26 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.286 1124 5.11 %
Rwork0.2452 --
obs0.2473 21992 95.43 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.75→47.492 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8864 3014 5 0 11883
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00312427
X-RAY DIFFRACTIONf_angle_d0.53917430
X-RAY DIFFRACTIONf_dihedral_angle_d24.3125076
X-RAY DIFFRACTIONf_chiral_restr0.0361932
X-RAY DIFFRACTIONf_plane_restr0.0031752
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.7502-3.92080.37051160.36332091X-RAY DIFFRACTION78
3.9208-4.12750.36521230.34242380X-RAY DIFFRACTION89
4.1275-4.38590.35691610.28852672X-RAY DIFFRACTION99
4.3859-4.72420.29861470.26972705X-RAY DIFFRACTION100
4.7242-5.19910.27321570.23642682X-RAY DIFFRACTION100
5.1991-5.95020.2961320.24592725X-RAY DIFFRACTION99
5.9502-7.49190.27591490.24562759X-RAY DIFFRACTION100
7.4919-47.49590.24041390.19562854X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: -27.1941 Å / Origin y: -24.6263 Å / Origin z: -40.1752 Å
111213212223313233
T0.8632 Å2-0.0364 Å20.1028 Å2-0.8412 Å2-0.042 Å2--0.9143 Å2
L1.5016 °20.182 °20.6832 °2-0.9886 °20.2991 °2--1.0598 °2
S0.0522 Å °-0.0063 Å °0.1177 Å °0.0095 Å °-0.1182 Å °0.2994 Å °-0.0288 Å °-0.139 Å °0.0437 Å °
Refinement TLS groupSelection details: all

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