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- PDB-8y0a: Crystal structure of LbCas12a in complex with crRNA and 18nt targ... -

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Basic information

Entry
Database: PDB / ID: 8y0a
TitleCrystal structure of LbCas12a in complex with crRNA and 18nt target DNA
Components
  • DNA (27-MER)
  • DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*TP*T)-3')
  • LbCas12a
  • RNA (38-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: 3.51 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 (38-MER)
C: DNA (27-MER)
D: DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*TP*T)-3')
E: LbCas12a
F: RNA (38-MER)
G: DNA (27-MER)
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)336,77812
Polymers336,6818
Non-polymers974
Water28816
1
A: LbCas12a
B: RNA (38-MER)
C: DNA (27-MER)
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)168,3896
Polymers168,3414
Non-polymers492
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16310 Å2
ΔGint-122 kcal/mol
Surface area64390 Å2
MethodPISA
2
E: LbCas12a
F: RNA (38-MER)
G: DNA (27-MER)
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)168,3896
Polymers168,3414
Non-polymers492
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16670 Å2
ΔGint-122 kcal/mol
Surface area63630 Å2
MethodPISA
Unit cell
Length a, b, c (Å)121.777, 142.673, 209.126
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 222 or (resid 223...AA1 - 10761 - 1076
121ASNASNVALVAL(chain 'A' and (resid 1 through 222 or (resid 223...AA1081 - 12261081 - 1226
211METMETARGARG(chain 'E' and (resid 1 through 384 or (resid 385...EE1 - 10761 - 1076
221ASNASNVALVAL(chain 'E' and (resid 1 through 384 or (resid 385...EE1081 - 12261081 - 1226
112AACCchain 'B'BB-20 - 171 - 38
212AACCchain 'F'FF-20 - 171 - 38
113DGDGDGDGchain 'C'CC-17 - 91 - 27
213DGDGDGDGchain 'G'GG-17 - 91 - 27
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 (27-MER)


Mass: 8357.384 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 (38-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, 20 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 16 / 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: 2.7 Å3/Da / Density % sol: 54.41 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.1875 mM lithium sulfate, 75 mM sodium phosphate dibasic, 50 mM citric acid, 16% PEG 1000

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Data collection

DiffractionMean temperature: 289 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: 3.51→50 Å / Num. obs: 46071 / % possible obs: 100 % / Redundancy: 11.3 % / Biso Wilson estimate: 48.66 Å2 / Rpim(I) all: 0.113 / Net I/σ(I): 8
Reflection shellResolution: 3.51→3.58 Å / Redundancy: 10.7 % / Mean I/σ(I) obs: 2.45 / Num. unique obs: 2289 / Rpim(I) all: 0.29 / % 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: 5XUZ

5xuz


Resolution: 3.51→25.87 Å / SU ML: 0.4102 / Cross valid method: FREE R-VALUE / σ(F): 2.03 / Phase error: 31.7435
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2938 2209 4.98 %
Rwork0.2376 42106 -
obs0.2404 44315 96.23 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 54.84 Å2
Refinement stepCycle: LAST / Resolution: 3.51→25.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19922 3160 4 16 23102
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.003823874
X-RAY DIFFRACTIONf_angle_d0.69632854
X-RAY DIFFRACTIONf_chiral_restr0.04263606
X-RAY DIFFRACTIONf_plane_restr0.0043668
X-RAY DIFFRACTIONf_dihedral_angle_d21.96139244
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.51-3.590.3495920.28571759X-RAY DIFFRACTION65.2
3.59-3.670.34341250.26692263X-RAY DIFFRACTION84.03
3.67-3.760.33221400.27432578X-RAY DIFFRACTION96.08
3.76-3.860.33691320.26322661X-RAY DIFFRACTION98.59
3.86-3.980.32671520.25622681X-RAY DIFFRACTION99.4
3.98-4.110.30781450.25212687X-RAY DIFFRACTION99.82
4.11-4.250.2361290.23222743X-RAY DIFFRACTION99.9
4.25-4.420.27231300.22342690X-RAY DIFFRACTION99.86
4.42-4.620.27221370.22132736X-RAY DIFFRACTION99.97
4.62-4.860.27781490.21582730X-RAY DIFFRACTION99.9
4.86-5.170.27781400.22652720X-RAY DIFFRACTION99.93
5.17-5.560.30591520.22882719X-RAY DIFFRACTION99.79
5.56-6.110.34161350.23532765X-RAY DIFFRACTION99.9
6.11-6.980.27621470.25152748X-RAY DIFFRACTION99.83
6.98-8.740.26371450.22062809X-RAY DIFFRACTION99.73
8.74-25.870.26151590.21082817X-RAY DIFFRACTION97.38

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