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- PDB-8y09: Crystal structure of LbCas12a in complex with crRNA and 15nt targ... -

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Basic information

Entry
Database: PDB / ID: 8y09
TitleCrystal structure of LbCas12a in complex with crRNA and 15nt target DNA
Components
  • DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*TP*T)-3')
  • DNA (5'-D(P*CP*TP*TP*TP*AP*CP*TP*GP*GP*AP*TP*GP*CP*GP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')
  • LbCas12a
  • RNA (34-MER)
KeywordsRNA BINDING PROTEIN / CRISPR / Cas12a / crRNA / Complex
Function / homology
Function and homology information


: / 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) / LbCas12a
Similarity search - Component
Biological speciesLachnospiraceae bacterium ND2006 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.87 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
A: LbCas12a
B: RNA (34-MER)
C: DNA (5'-D(P*CP*TP*TP*TP*AP*CP*TP*GP*GP*AP*TP*GP*CP*GP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')
D: DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*TP*T)-3')
E: LbCas12a
F: RNA (34-MER)
G: DNA (5'-D(P*CP*TP*TP*TP*AP*CP*TP*GP*GP*AP*TP*GP*CP*GP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')
H: DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)334,90113
Polymers334,8668
Non-polymers355
Water18010
1
A: LbCas12a
B: RNA (34-MER)
C: DNA (5'-D(P*CP*TP*TP*TP*AP*CP*TP*GP*GP*AP*TP*GP*CP*GP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')
D: DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,4547
Polymers167,4334
Non-polymers213
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13700 Å2
ΔGint-111 kcal/mol
Surface area64130 Å2
MethodPISA
2
E: LbCas12a
F: RNA (34-MER)
G: DNA (5'-D(P*CP*TP*TP*TP*AP*CP*TP*GP*GP*AP*TP*GP*CP*GP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')
H: DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,4476
Polymers167,4334
Non-polymers142
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13210 Å2
ΔGint-103 kcal/mol
Surface area64380 Å2
MethodPISA
Unit cell
Length a, b, c (Å)122.149, 143.401, 203.528
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
12
22
13
23
14
24

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111METMETARGARG(chain 'A' and (resid 1 through 1075 or (resid 1084...AA1 - 2841 - 284
121GLYGLYPHEPHE(chain 'A' and (resid 1 through 1075 or (resid 1084...AA291 - 1075291 - 1075
131PHEPHEVALVAL(chain 'A' and (resid 1 through 1075 or (resid 1084...AA1084 - 12261084 - 1226
211METMETARGARG(chain 'E' and (resid 1 through 112 or (resid 113...EE1 - 2841 - 284
221GLYGLYPHEPHE(chain 'E' and (resid 1 through 112 or (resid 113...EE291 - 1075291 - 1075
231PHEPHEVALVAL(chain 'E' and (resid 1 through 112 or (resid 113...EE1084 - 12261084 - 1226
112AAGGchain 'B'BB-20 - 131 - 34
212AAGGchain 'F'FF-20 - 131 - 34
113DCDCDGDGchain 'C'CC-13 - 92 - 24
213DCDCDGDGchain 'G'GG-13 - 92 - 24
114DCDCDTDTchain 'D'DD-9 - 11 - 11
214DCDCDTDTchain 'H'HH-9 - 11 - 11

NCS ensembles :
ID
1
2
3
4

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Components

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DNA chain , 2 types, 4 molecules CGDH

#3: DNA chain DNA (5'-D(P*CP*TP*TP*TP*AP*CP*TP*GP*GP*AP*TP*GP*CP*GP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')


Mass: 7449.814 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)
#4: DNA chain DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*TP*T)-3')


Mass: 3290.157 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)

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Protein / RNA chain , 2 types, 4 molecules AEBF

#1: Protein LbCas12a


Mass: 143888.359 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A5S8WF58
#2: RNA chain RNA (34-MER)


Mass: 12804.658 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)

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Non-polymers , 2 types, 15 molecules

#5: Chemical
ChemComp-LI / LITHIUM ION


Mass: 6.941 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Li
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 10 / 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: 2.66 Å3/Da / Density % sol: 53.79 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.2 M Li2SO4, 30 mM Na2HPO4, 43.75 mM citric acid, 13% PEG 1000

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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: Mar 15, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 2.85→50 Å / Num. obs: 81224 / % possible obs: 99.7 % / Redundancy: 8 % / Biso Wilson estimate: 50.18 Å2 / Rpim(I) all: 0.1 / Net I/σ(I): 7.25
Reflection shellResolution: 2.85→2.9 Å / Redundancy: 6.5 % / Num. unique obs: 4005 / Rpim(I) all: 0.402 / % possible all: 99.5

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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: 5XUZZ

Resolution: 2.87→49.28 Å / SU ML: 0.3482 / Cross valid method: FREE R-VALUE / σ(F): 1.4 / Phase error: 27.1509
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.267 3462 4.97 %
Rwork0.2043 66252 -
obs0.2074 69714 84.55 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 50.11 Å2
Refinement stepCycle: LAST / Resolution: 2.87→49.28 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19913 2824 5 10 22752
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.011323486
X-RAY DIFFRACTIONf_angle_d1.390432229
X-RAY DIFFRACTIONf_chiral_restr0.06963515
X-RAY DIFFRACTIONf_plane_restr0.00823643
X-RAY DIFFRACTIONf_dihedral_angle_d24.89649116
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.87-2.910.319590.3615206X-RAY DIFFRACTION6.57
2.91-2.950.4703330.3138710X-RAY DIFFRACTION22.79
2.95-2.990.3601520.30111075X-RAY DIFFRACTION34.68
2.99-3.040.411660.29721556X-RAY DIFFRACTION49.79
3.04-3.090.29341130.29112051X-RAY DIFFRACTION66.75
3.09-3.140.32751280.27222483X-RAY DIFFRACTION79.92
3.14-3.20.33311390.26472675X-RAY DIFFRACTION86.03
3.2-3.260.33911590.25522762X-RAY DIFFRACTION89.6
3.26-3.330.30921440.2412852X-RAY DIFFRACTION91.93
3.33-3.40.30541690.24082926X-RAY DIFFRACTION94.74
3.4-3.480.28181680.23432975X-RAY DIFFRACTION95.97
3.48-3.570.31021860.22853003X-RAY DIFFRACTION97.67
3.57-3.660.31781560.21593077X-RAY DIFFRACTION98.24
3.66-3.770.26721760.20213069X-RAY DIFFRACTION99.11
3.77-3.890.26561420.19943103X-RAY DIFFRACTION99.14
3.89-4.030.25151680.19033110X-RAY DIFFRACTION99.57
4.03-4.190.24731580.1793131X-RAY DIFFRACTION99.73
4.19-4.380.24221650.17993142X-RAY DIFFRACTION99.82
4.38-4.610.23421470.16283145X-RAY DIFFRACTION99.94
4.61-4.90.23981690.1593134X-RAY DIFFRACTION100
4.9-5.280.23611660.1743165X-RAY DIFFRACTION100
5.28-5.810.25481550.17423184X-RAY DIFFRACTION99.94
5.81-6.650.25271560.19773179X-RAY DIFFRACTION99.97
6.65-8.370.22061730.1943224X-RAY DIFFRACTION100
8.38-49.280.23551650.20393315X-RAY DIFFRACTION98.11

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