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- PDB-5u34: Crystal structure of AacC2c1-sgRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 5u34
TitleCrystal structure of AacC2c1-sgRNA binary complex
Components
  • CRISPR-associated endonuclease C2c1
  • sgRNA
KeywordsHYDROLASE/RNA / 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-RNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / 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
RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas12b
Similarity search - Component
Biological speciesAlicyclobacillus acidoterrestris (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.255 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
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
Revision 1.4Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2
Revision 1.5Oct 30, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease C2c1
B: sgRNA


Theoretical massNumber of molelcules
Total (without water)168,0192
Polymers168,0192
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8210 Å2
ΔGint-63 kcal/mol
Surface area59310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)83.471, 128.198, 155.788
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein CRISPR-associated endonuclease C2c1 / AacC2c1


Mass: 131835.047 Da / Num. of mol.: 1 / Fragment: CRISPR-associated endonuclease AacC2c1 / Mutation: E848A
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)
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: 2.48 Å3/Da / Density % sol: 50.41 %
Crystal growTemperature: 293 K / Method: evaporation / pH: 7.2
Details: 0.2 M lithium sulfate, 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: Jun 25, 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: 3.25→50 Å / Num. obs: 26140 / % possible obs: 99.2 % / Redundancy: 5.9 % / Biso Wilson estimate: 106.74 Å2 / CC1/2: 0.99 / Net I/σ(I): 10.2
Reflection shellResolution: 3.25→3.48 Å / Redundancy: 5.7 % / Mean I/σ(I) obs: 1 / CC1/2: 0.318 / % possible all: 95.6

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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: 3.255→48.33 Å / SU ML: 0.63 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 38.19 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3235 1278 4.89 %
Rwork0.2695 --
obs0.2719 26140 97.2 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.255→48.33 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7882 1680 0 0 9562
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0019927
X-RAY DIFFRACTIONf_angle_d0.40113762
X-RAY DIFFRACTIONf_dihedral_angle_d18.6525792
X-RAY DIFFRACTIONf_chiral_restr0.0331524
X-RAY DIFFRACTIONf_plane_restr0.0031492
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2545-3.38480.45141120.38742088X-RAY DIFFRACTION75
3.3848-3.53880.4791300.35532819X-RAY DIFFRACTION100
3.5388-3.72530.35661440.30422776X-RAY DIFFRACTION100
3.7253-3.95860.33231440.28342825X-RAY DIFFRACTION100
3.9586-4.26410.32971410.26292797X-RAY DIFFRACTION100
4.2641-4.69290.29811510.23332839X-RAY DIFFRACTION100
4.6929-5.37120.27361460.24172838X-RAY DIFFRACTION100
5.3712-6.76420.33441540.28622886X-RAY DIFFRACTION100
6.7642-48.33560.30641560.25512994X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: 16.8668 Å / Origin y: -23.3861 Å / Origin z: -26.9779 Å
111213212223313233
T0.5314 Å20.1194 Å2-0.1081 Å2-0.6559 Å20.0328 Å2--0.5894 Å2
L1.1238 °20.5996 °20.0223 °2-2.4582 °2-0.0375 °2--1.3643 °2
S-0.0396 Å °-0.0407 Å °-0.0028 Å °-0.0611 Å °-0.1119 Å °-0.1159 Å °0.0772 Å °0.367 Å °0.1534 Å °
Refinement TLS groupSelection details: all

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