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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 / Acting on Ester Bonds / RNA binding / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Specimen sourceLeptotrichia buccalis (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / 3.086 Å resolution
AuthorsLiu, L. / Li, X. / Li, Z. / Wang, Y.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 30, 2017 / Release: Sep 13, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Sep 13, 2017Structure modelrepositoryInitial release
1.1Oct 18, 2017Structure modelAuthor supporting evidencepdbx_audit_support_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
Polyers336,3546
Non-polymers00
Water21612
1
A: Uncharacterized protein
C: RNA (59-MER)
D: RNA (30-MER)


Theoretical massNumber of molelcules
Total (without water)168,1773
Polyers168,1773
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)17080
ΔGint (kcal/M)-124
Surface area (Å2)61120
MethodPISA
2
E: RNA (59-MER)
F: RNA (30-MER)
B: Uncharacterized protein


Theoretical massNumber of molelcules
Total (without water)168,1773
Polyers168,1773
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)16210
ΔGint (kcal/M)-127
Surface area (Å2)61480
MethodPISA
Unit cell
γ
α
β
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-MP 1 21 1

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Components

#1: Protein/peptide Uncharacterized protein


Mass: 139627.047 Da / Num. of mol.: 2 / Mutation: R1048A, H1053A / 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: (synth.) synthetic construct (others)
#3: RNA chain RNA (30-MER)


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


Mass: 18.015 Da / Num. of mol.: 12 / Formula: H2O / Water

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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 / Density percent sol: 54.51 %
Crystal growTemp: 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 kelvins
SourceSource: SYNCHROTRON / Type: SSRF BEAMLINE BL17U1 / Synchrotron site: SSRF / Beamline: BL17U1 / Wavelength: 0.9785
DetectorType: RAYONIX MX-225 / Detector: CCD / Collection date: Mar 21, 2017
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9785 Å / Relative weight: 1
ReflectionD resolution high: 3.08 Å / D resolution low: 5 Å / Number obs: 61255 / Rmerge I obs: 0.185 / NetI over sigmaI: 14 / Redundancy: 15 % / Percent possible obs: 98.6
Reflection shellRmerge I obs: 0.558 / Highest resolution: 3.08 Å / Lowest resolution: 3.13 Å / MeanI over sigI obs: 3.5 / Redundancy: 1 % / Percent possible all: 98.6

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155)refinement
HKL-3000data reduction
HKL-3000data scaling
PHASERphasing
RefineMethod to determine structure: SAD / Overall SU ML: 0.44 / Cross valid method: FREE R-VALUE / Sigma F: 1.52 / Overall phase error: 22.72 / Stereochemistry target values: ML
Solvent computationSolvent shrinkage radii: 0.9 Å / Solvent vdw probe radii: 1.11 Å / Solvent model details: FLAT BULK SOLVENT MODEL
Least-squares processR factor R free: 0.2437 / R factor R work: 0.2185 / R factor obs: 0.2197 / Highest resolution: 3.086 Å / Lowest resolution: 48.179 Å / Number reflection R free: 2881 / Number reflection obs: 61255 / Percent reflection R free: 4.7 / Percent reflection obs: 95.1
Refine hist #LASTHighest resolution: 3.086 Å / Lowest resolution: 48.179 Å
Number of atoms included #LASTProtein: 18100 / Nucleic acid: 3726 / Ligand: 0 / Solvent: 12 / Total: 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
Refine LS shell

Refine ID: X-RAY DIFFRACTION

Highest resolutionR factor R freeR factor R workLowest resolutionNumber reflection R freeNumber reflection R workPercent reflection obs
3.08590.35710.30943.1365114210773.00
3.13650.33340.30463.1906123240683.00
3.19060.32230.30503.2486148250487.00
3.24860.35460.29263.3111125265591.00
3.31110.31490.27943.3786127272994.00
3.37860.30320.28283.4521115276494.00
3.45210.30660.27623.5323155276896.00
3.53230.27880.25683.6207163279196.00
3.62070.30710.23983.7185138281297.00
3.71850.22840.22463.8279119285697.00
3.82790.22600.22053.9514170279597.00
3.95140.21730.20474.0925139286398.00
4.09250.23780.19814.2563150284098.00
4.25630.22290.18404.4499112290198.00
4.44990.18670.18434.6843162289499.00
4.68430.20050.17524.9775129291799.00
4.97750.21810.18335.36141542915100.00
5.36140.22150.19995.90011202926100.00
5.90010.22340.20876.75181512950100.00
6.75180.21160.19368.49901322957100.00
8.49900.20070.191348.18481353024100.00

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