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- PDB-8y0b: Crystal structure of FnCas12a in complex with pre-crRNA and 12nt ... -

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Basic information

Entry
Database: PDB / ID: 8y0b
TitleCrystal structure of FnCas12a in complex with pre-crRNA and 12nt target DNA
Components
  • CRISPR-associated endonuclease Cas12a
  • DNA (5'-D(P*AP*AP*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*AP*AP*AP*GP*GP*AP*CP*T)-3')
  • DNA (5'-D(P*AP*GP*TP*CP*CP*TP*TP*TP*AP*GP*AP*TP*A)-3')
  • RNA (53-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: 2.3 Å
AuthorsLin, X. / Chen, 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 (53-MER)
C: DNA (5'-D(P*AP*AP*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*AP*AP*AP*GP*GP*AP*CP*T)-3')
D: DNA (5'-D(P*AP*GP*TP*CP*CP*TP*TP*TP*AP*GP*AP*TP*A)-3')
A: CRISPR-associated endonuclease Cas12a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,6806
Polymers182,6314
Non-polymers492
Water10,233568
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16500 Å2
ΔGint-126 kcal/mol
Surface area68690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)124.729, 124.729, 267.756
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 (5'-D(P*AP*AP*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*AP*AP*AP*GP*GP*AP*CP*T)-3')


Mass: 6430.199 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*GP*AP*TP*A)-3')


Mass: 5529.614 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 (53-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, 570 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.: 568 / 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: 56.86 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.05 M MES, pH 6.0, 0.15 M sodium formate, 15% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL02U1 / Wavelength: 0.98322 Å
DetectorType: DECTRIS EIGER2 S 9M / Detector: PIXEL / Date: Nov 25, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98322 Å / Relative weight: 1
ReflectionResolution: 2.3→88.2 Å / Num. obs: 91977 / % possible obs: 97.2 % / Redundancy: 13.8 % / Biso Wilson estimate: 39.94 Å2 / Rpim(I) all: 0.044 / Net I/σ(I): 15.9
Reflection shellResolution: 2.3→2.42 Å / Mean I/σ(I) obs: 2.3 / Num. unique obs: 13638 / Rpim(I) all: 0.395 / % possible all: 100

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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: 2.3→49.21 Å / SU ML: 0.2614 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.1358
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2475 4587 5 %
Rwork0.2028 87229 -
obs0.205 91816 97.13 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 53.78 Å2
Refinement stepCycle: LAST / Resolution: 2.3→49.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10400 1816 2 568 12786
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.008612635
X-RAY DIFFRACTIONf_angle_d1.084317386
X-RAY DIFFRACTIONf_chiral_restr0.05641923
X-RAY DIFFRACTIONf_plane_restr0.00561919
X-RAY DIFFRACTIONf_dihedral_angle_d23.6654994
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.330.29061800.26422967X-RAY DIFFRACTION100
2.33-2.350.32471520.25672927X-RAY DIFFRACTION100
2.35-2.380.29541690.26032945X-RAY DIFFRACTION100
2.38-2.410.29091680.25452933X-RAY DIFFRACTION100
2.41-2.440.28351400.24452962X-RAY DIFFRACTION100
2.44-2.480.29041570.24542929X-RAY DIFFRACTION100
2.48-2.510.2991550.24042957X-RAY DIFFRACTION100
2.51-2.550.28291540.23982968X-RAY DIFFRACTION100
2.55-2.590.29671550.24062953X-RAY DIFFRACTION100
2.59-2.630.29271690.24392952X-RAY DIFFRACTION100
2.63-2.660.3303860.24942088X-RAY DIFFRACTION99.82
2.69-2.730.33461160.25022430X-RAY DIFFRACTION98.72
2.73-2.780.28091600.2312954X-RAY DIFFRACTION100
2.78-2.830.26931780.23882956X-RAY DIFFRACTION100
2.83-2.90.2681630.23212954X-RAY DIFFRACTION99.97
2.9-2.960.30441530.23292971X-RAY DIFFRACTION100
2.96-3.040.26681630.22442977X-RAY DIFFRACTION100
3.04-3.120.27841550.22462967X-RAY DIFFRACTION99.97
3.12-3.210.2721700.22242979X-RAY DIFFRACTION100
3.21-3.320.27611620.21412976X-RAY DIFFRACTION100
3.32-3.420.24881220.20142674X-RAY DIFFRACTION99.86
3.47-3.570.23211260.20162209X-RAY DIFFRACTION98.4
3.57-3.730.25211360.19992994X-RAY DIFFRACTION100
3.73-3.930.25261490.18343032X-RAY DIFFRACTION100
3.93-4.180.2071570.1733021X-RAY DIFFRACTION99.97
4.18-4.50.20751450.16623047X-RAY DIFFRACTION100
4.5-4.950.22191560.16243061X-RAY DIFFRACTION100
4.95-5.670.2221700.17323060X-RAY DIFFRACTION100
5.67-7.140.23411570.19873119X-RAY DIFFRACTION100
7.14-49.210.18231640.18143267X-RAY DIFFRACTION98.99

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