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- PDB-5xwy: Electron cryo-microscopy structure of LbuCas13a-crRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 5xwy
TitleElectron cryo-microscopy structure of LbuCas13a-crRNA binary complex
DescriptorUncharacterized protein/RNA Complex
KeywordsRNA BINDING PROTEIN/RNA / Cas13a / CRISPR / RNA BINDING PROTEIN-RNA COMPLEX
Specimen sourceLeptotrichia buccalis (strain atcc 14201 / dsm 1135 / jcm 12969 / nctc 10249 / c-1013-b) / bacteria
Synthetic construct
MethodElectron microscopy (3.2 Å resolution / Particle / Single particle)
AuthorsZhang, X. / Wang, Y. / Ma, J. / Liu, L. / Li, X. / Li, Z. / You, L. / Wang, J. / Wang, M.
CitationCell, 2017, 170, 714-726.e10

Cell, 2017, 170, 714-726.e10 StrPapers
The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a.
Liang Liu / Xueyan Li / Jun Ma / Zongqiang Li / Lilan You / Jiuyu Wang / Min Wang / Xinzheng Zhang / Yanli Wang

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 30, 2017 / Release: Sep 13, 2017

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Assembly

Deposited unit
A: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
B: RNA (59-MER)


Theoretical massNumber of molelcules
Total (without water)157,4252
Polyers157,4252
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)11180
ΔGint (kcal/M)-71
Surface area (Å2)56350

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Components

#1: Polypeptide(L)A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a


Mass: 138555.156 Da / Num. of mol.: 1 / Mutation: R1048A, H1053A
Source: (gene. exp.) Leptotrichia buccalis (strain atcc 14201 / dsm 1135 / jcm 12969 / nctc 10249 / c-1013-b) / bacteria
References: UniProt: C7NBY4
#2: RNA chainRNA (59-MER)


Mass: 18869.348 Da / Num. of mol.: 1 / Source: (synth.) Synthetic construct

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: LbuCas13a-crRNA binary complex / Type: COMPLEX / Entity ID: 1, 2 / Source: RECOMBINANT
Source (natural)Organism: Leptotrichia buccalis / Strain: ATCC 14201
Source (recombinant)Organism: Escherichia coli
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_2411: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 238758 / Symmetry type: POINT
Refine
Refine IDHighest resolution
13.20
ELECTRON MICROSCOPY
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01010528
ELECTRON MICROSCOPYf_angle_d1.20114384
ELECTRON MICROSCOPYf_dihedral_angle_d11.7718615
ELECTRON MICROSCOPYf_chiral_restr0.0631619
ELECTRON MICROSCOPYf_plane_restr0.0051642

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