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- PDB-5xwp: Crystal structure of LbuCas13a-crRNA-target RNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 5xwp
TitleCrystal structure of LbuCas13a-crRNA-target RNA ternary complex
Components
  • RNA (30-MER)
  • RNA (59-MER)
  • Uncharacterized protein
KeywordsRNA BINDING PROTEIN/RNA / LbuCas13a / C2c2 / crRNA / target RNA / RNA BINDING PROTEIN-RNA complex
Function / homologydefense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding / RNA / RNA (> 10) / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Biological speciesLeptotrichia buccalis (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3.086 Å
AuthorsLiu, L. / Li, X. / Li, Z. / Wang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2014CB910102 China
CitationJournal: Cell / Year: 2017
Title: The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a.
Authors: Liang Liu / Xueyan Li / Jun Ma / Zongqiang Li / Lilan You / Jiuyu Wang / Min Wang / Xinzheng Zhang / Yanli Wang /
Abstract: Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a ...Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.
History
DepositionJun 30, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 13, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Uncharacterized protein
C: RNA (59-MER)
D: RNA (30-MER)
E: RNA (59-MER)
F: RNA (30-MER)
B: Uncharacterized protein


Theoretical massNumber of molelcules
Total (without water)336,3546
Polymers336,3546
Non-polymers00
Water21612
1
A: Uncharacterized protein
C: RNA (59-MER)
D: RNA (30-MER)


Theoretical massNumber of molelcules
Total (without water)168,1773
Polymers168,1773
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17080 Å2
ΔGint-124 kcal/mol
Surface area61120 Å2
MethodPISA
2
E: RNA (59-MER)
F: RNA (30-MER)
B: Uncharacterized protein


Theoretical massNumber of molelcules
Total (without water)168,1773
Polymers168,1773
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16210 Å2
ΔGint-127 kcal/mol
Surface area61480 Å2
MethodPISA
Unit cell
Length a, b, c (Å)95.576, 132.870, 139.960
Angle α, β, γ (deg.)90.00, 90.80, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Uncharacterized protein


Mass: 139627.047 Da / Num. of mol.: 2 / Mutation: R1048A, H1053A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Leptotrichia buccalis (bacteria)
Strain: ATCC 14201 / DSM 1135 / JCM 12969 / NCTC 10249 / C-1013-b
Gene: Lebu_1799 / Production host: Escherichia coli K-12 (bacteria) / Strain (production host): K-12 / References: UniProt: C7NBY4
#2: RNA chain RNA (59-MER)


Mass: 18869.348 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: RNA chain RNA (30-MER)


Mass: 9680.769 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Formula: H2O

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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: 54.51 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop / pH: 7.5 / Details: 0.1 M HEPES, pH 7.5, 0.1 M L-Proline, 8% PEG 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.9785 Å
DetectorType: RAYONIX MX-225 / Detector: CCD / Date: Mar 21, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9785 Å / Relative weight: 1
ReflectionResolution: 3.08→50 Å / Num. obs: 61255 / % possible obs: 98.6 % / Redundancy: 15 % / Rmerge(I) obs: 0.185 / Net I/σ(I): 14
Reflection shellResolution: 3.08→3.13 Å / Redundancy: 10 % / Rmerge(I) obs: 0.558 / Mean I/σ(I) obs: 3.5 / % possible all: 98.6

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155)refinement
HKL-3000data reduction
HKL-3000data scaling
PHASERphasing
RefinementMethod to determine structure: SAD / Resolution: 3.086→48.179 Å / SU ML: 0.44 / Cross valid method: FREE R-VALUE / σ(F): 1.52 / Phase error: 22.72 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2437 2881 4.7 %
Rwork0.2185 --
obs0.2197 61255 95.1 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.086→48.179 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms18100 3726 0 12 21838
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01322568
X-RAY DIFFRACTIONf_angle_d1.59431192
X-RAY DIFFRACTIONf_dihedral_angle_d24.7539042
X-RAY DIFFRACTIONf_chiral_restr0.1043580
X-RAY DIFFRACTIONf_plane_restr0.0073343
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.0859-3.13650.35711140.30942107X-RAY DIFFRACTION73
3.1365-3.19060.33341230.30462406X-RAY DIFFRACTION83
3.1906-3.24860.32231480.3052504X-RAY DIFFRACTION87
3.2486-3.31110.35461250.29262655X-RAY DIFFRACTION91
3.3111-3.37860.31491270.27942729X-RAY DIFFRACTION94
3.3786-3.45210.30321150.28282764X-RAY DIFFRACTION94
3.4521-3.53230.30661550.27622768X-RAY DIFFRACTION96
3.5323-3.62070.27881630.25682791X-RAY DIFFRACTION96
3.6207-3.71850.30711380.23982812X-RAY DIFFRACTION97
3.7185-3.82790.22841190.22462856X-RAY DIFFRACTION97
3.8279-3.95140.2261700.22052795X-RAY DIFFRACTION97
3.9514-4.09250.21731390.20472863X-RAY DIFFRACTION98
4.0925-4.25630.23781500.19812840X-RAY DIFFRACTION98
4.2563-4.44990.22291120.1842901X-RAY DIFFRACTION98
4.4499-4.68430.18671620.18432894X-RAY DIFFRACTION99
4.6843-4.97750.20051290.17522917X-RAY DIFFRACTION99
4.9775-5.36140.21811540.18332915X-RAY DIFFRACTION100
5.3614-5.90010.22151200.19992926X-RAY DIFFRACTION100
5.9001-6.75180.22341510.20872950X-RAY DIFFRACTION100
6.7518-8.4990.21161320.19362957X-RAY DIFFRACTION100
8.499-48.18480.20071350.19133024X-RAY DIFFRACTION100

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