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- PDB-8y0c: Crystal structure of FnCas12a in complex with pre-crRNA and 18nt ... -

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Basic information

Entry
Database: PDB / ID: 8y0c
TitleCrystal structure of FnCas12a in complex with pre-crRNA and 18nt target DNA
Components
  • CRISPR-associated endonuclease Cas12a
  • DNA (27-MER)
  • DNA (5'-D(P*AP*GP*TP*CP*CP*TP*TP*TP*AP*CP*T)-3')
  • RNA (42-MER)
KeywordsRNA BINDING PROTEIN / CRISPR / Cas12a / crRNA / Complex
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
: / CRISPR-associated endonuclease Cpf1 PI domain / : / CRISPR-associated endonuclease Cpf1 REC2 domain / CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida U112 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.45 Å
AuthorsChen, J. / Liu, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32022047 China
CitationJournal: Nucleic Acids Res. / Year: 2025
Title: DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems.
Authors: Chen, J. / Lin, X. / Xiang, W. / Chen, Y. / Zhao, Y. / Huang, L. / Liu, L.
History
DepositionJan 22, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: RNA (42-MER)
C: DNA (27-MER)
D: DNA (5'-D(P*AP*GP*TP*CP*CP*TP*TP*TP*AP*CP*T)-3')
A: CRISPR-associated endonuclease Cas12a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,2686
Polymers182,2204
Non-polymers492
Water362
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16640 Å2
ΔGint-121 kcal/mol
Surface area67030 Å2
MethodPISA
Unit cell
Length a, b, c (Å)124.283, 124.283, 268.523
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

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DNA chain , 2 types, 2 molecules CD

#2: DNA chain DNA (27-MER)


Mass: 8249.358 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)
#3: DNA chain DNA (5'-D(P*AP*GP*TP*CP*CP*TP*TP*TP*AP*CP*T)-3')


Mass: 3299.171 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)

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RNA chain / Protein , 2 types, 2 molecules BA

#1: RNA chain RNA (42-MER)


Mass: 18587.984 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)
#4: Protein CRISPR-associated endonuclease Cas12a / CRISPR-associated endonuclease Cpf1 / FnCas12a / FnCpf1


Mass: 152083.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. novicida U112 (bacteria)
Gene: cas12a, cpf1, FTN_1397 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0Q7Q2, deoxyribonuclease I, Bacillus subtilis ribonuclease

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationN

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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: 2.85 Å3/Da / Density % sol: 56.77 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.05 M MES, pH 6.0, 0.15 M Sodium formate, 17% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL18U1 / Wavelength: 0.97915 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 22, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 3.45→50 Å / Num. obs: 28418 / % possible obs: 99.8 % / Redundancy: 8.1 % / Biso Wilson estimate: 48.2 Å2 / Rpim(I) all: 0.102 / Net I/σ(I): 6
Reflection shellResolution: 3.45→3.51 Å / Redundancy: 6.4 % / Mean I/σ(I) obs: 2.5 / Num. unique obs: 1362 / Rpim(I) all: 0.279 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
HKL-30007.21data scaling
HKL-30007.21data reduction
Coot0.7.2.1model building
PHENIX1.17.1_3660phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5MGA

5mga


Resolution: 3.45→47.22 Å / SU ML: 0.4733 / Cross valid method: FREE R-VALUE / σ(F): 1.41 / Phase error: 24.7791
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2853 1234 4.89 %
Rwork0.2159 24017 -
obs0.2193 25251 88.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 50.39 Å2
Refinement stepCycle: LAST / Resolution: 3.45→47.22 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10393 1661 2 2 12058
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.011212451
X-RAY DIFFRACTIONf_angle_d1.34117093
X-RAY DIFFRACTIONf_chiral_restr0.06231881
X-RAY DIFFRACTIONf_plane_restr0.0071904
X-RAY DIFFRACTIONf_dihedral_angle_d19.66242227
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.45-3.590.3497350.2468806X-RAY DIFFRACTION27.52
3.59-3.760.30431170.24932207X-RAY DIFFRACTION74.82
3.76-3.950.31161650.23162808X-RAY DIFFRACTION96.46
3.95-4.20.28761450.22552958X-RAY DIFFRACTION99.39
4.2-4.530.28511330.21382984X-RAY DIFFRACTION99.9
4.53-4.980.3061580.20972981X-RAY DIFFRACTION99.81
4.98-5.70.30271600.20243002X-RAY DIFFRACTION99.87
5.7-7.180.27361640.21353035X-RAY DIFFRACTION99.91
7.18-47.220.23231570.19823236X-RAY DIFFRACTION99.74

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