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- PDB-5vzl: cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex -

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Basic information

Entry
Database: PDB / ID: 5vzl
Titlecryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex
DescriptorCRISPR-associated endonuclease Cas9
phage anti-CRISPR AcrIIA4/RNA Complex
Keywordsanti-CRISPR / Cas9 / CRISPR / gene editing / on-target / off-target / cryo-EM
Specimen sourceStreptococcus pyogenes m1 gas / bacteria
unidentified phage / virus
MethodElectron microscopy (3.9 Å resolution / Particle / Single particle)
AuthorsJiang, F. / Liu, J.J. / Nogales, E. / Doudna, J.A.
CitationSci Adv, 2017, 3, e1701620-e1701620

Sci Adv, 2017, 3, e1701620-e1701620 Yorodumi Papers
Disabling Cas9 by an anti-CRISPR DNA mimic.
Jiyung Shin / Fuguo Jiang / Jun-Jie Liu / Nicolas L Bray / Benjamin J Rauch / Seung Hyun Baik / Eva Nogales / Joseph Bondy-Denomy / Jacob E Corn / Jennifer A Doudna

Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 29, 2017 / Release: Jul 26, 2017

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Assembly

Deposited unit
B: single guide RNA (116-MER)
A: CRISPR-associated endonuclease Cas9
C: phage anti-CRISPR AcrIIA4


Theoretical massNumber of molelcules
Total (without water)207,2483
Polyers207,2483
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)17050
ΔGint (kcal/M)-115
Surface area (Å2)84820

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Components

#1: RNA chainsingle guide RNA (116-MER)


Mass: 38292.645 Da / Num. of mol.: 1
Source: (gene. exp.) Streptococcus pyogenes m1 gas / bacteria
#2: Polypeptide(L)CRISPR-associated endonuclease Cas9


Mass: 158772.891 Da / Num. of mol.: 1
Source: (gene. exp.) Streptococcus pyogenes m1 gas / bacteria
References: UniProt: A0A0C6FZC2, EC: 3.1.-.-
#3: Polypeptide(L)phage anti-CRISPR AcrIIA4


Mass: 10182.073 Da / Num. of mol.: 1 / Source: (gene. exp.) unidentified phage / virus

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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

Component
IDNameTypeEntity IDParent IDSource
1the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4COMPLEX1,2,30RECOMBINANT
2Streptococcus pyogenes M1 GASCOMPLEX1,21RECOMBINANT
3unidentified phageCOMPLEX31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
21160490Streptococcus pyogenes M1 GAS
3238018unidentified phage
Source (recombinant)Organism: Escherichia coli
Buffer solutionpH: 8
SpecimenConc.: 1.25 mg/ml / Details: spyCas9, sgRNA and AcrIIA4 complex / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: C-flat 2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 kelvins
Details: blot for 4.5 seconds with force of 12 before plunging

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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 / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 4000 nm / Cs: 0.01 mm / C2 aperture diameter: 100 mm
Image recordingElectron dose: 46 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategoryDetailsImage processing ID
4Relion2.0CTF CORRECTIONCTFFIND41
11Relion2.0FINAL EULER ASSIGNMENT1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 185000 / Symmetry type: POINT
Atomic model building
IDRef protocolRef space
1AB INITIO MODELREAL
2RIGID BODY FITREAL
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614414
ELECTRON MICROSCOPYf_angle_d0.92720059
ELECTRON MICROSCOPYf_dihedral_angle_d12.1258452
ELECTRON MICROSCOPYf_chiral_restr0.0502356
ELECTRON MICROSCOPYf_plane_restr0.0062153

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