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- PDB-8h7q: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade at 3.8 ang... -

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Basic information

Entry
Database: PDB / ID: 8h7q
TitleCryo-EM structure of Synechocystis sp. PCC6714 Cascade at 3.8 angstrom resolution
Components
  • (CRISPR associated protein ...) x 3
  • CRISPR RNA
  • Non target DNA
  • Target DNA
KeywordsRNA BINDING PROTEIN / Type I-B / CRISPR-Cas / Cascade
Function / homology
Function and homology information


defense response to virus
Similarity search - Function
CRISPR type MYXAN-associated protein Cmx8 / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Uncharacterized protein / Fruiting body developmental protein R-like protein / Type I-MYXAN CRISPR-associated protein Cmx8
Similarity search - Component
Biological speciesSynechocystis sp. PCC 6714 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsXiao, Y. / Lu, M. / Yu, C. / Zhang, Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970547 China
Ministry of Science and Technology (MoST, China)2018YFA0902000 China
CitationJournal: To Be Published
Title: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade at 3.8 angstrom resolution
Authors: Xiao, Y. / Lu, M. / Yu, C. / Zhang, Y.
History
DepositionOct 20, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: CRISPR RNA
L: Target DNA
M: Non target DNA
B: CRISPR associated protein Cas8
C: CRISPR associated protein Cas8
E: CRISPR associated protein Cas8
F: CRISPR associated protein Cas8
G: CRISPR associated protein Cas8
H: CRISPR associated protein Cas8
I: CRISPR associated protein Cas8
J: CRISPR associated protein Cas11b
K: CRISPR associated protein Cas11b
N: CRISPR associated protein Cas11b
O: CRISPR associated protein Cas11b
A: CRISPR associated protein Cas5


Theoretical massNumber of molelcules
Total (without water)346,92015
Polymers346,92015
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 1 types, 1 molecules D

#1: RNA chain CRISPR RNA


Mass: 11380.655 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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DNA chain , 2 types, 2 molecules LM

#2: DNA chain Target DNA


Mass: 10847.038 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Non target DNA


Mass: 3422.260 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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CRISPR associated protein ... , 3 types, 12 molecules BCEFGHIJKNOA

#4: Protein
CRISPR associated protein Cas8 / Fruiting body developmental protein R-like protein


Mass: 33789.074 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714 / Gene: D082_50510
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A068N458
#5: Protein
CRISPR associated protein Cas11b / Type I-B CRISPR-associated protein Cas8b/Csh1


Mass: 14538.500 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714 / Gene: D082_50500
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A068N831
#6: Protein CRISPR associated protein Cas5 / Type I-MYXAN CRISPR-associated protein Cas5/Cmx5/DevS


Mass: 26592.424 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714 / Gene: D082_50520
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A068N1Y0

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Type I-B Cascade / Type: COMPLEX / Entity ID: #1-#3, #6, #4-#5 / Source: RECOMBINANT
Source (natural)Organism: Synechocystis sp. PCC 6714 (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2100 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175158 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00323569
ELECTRON MICROSCOPYf_angle_d0.83932275
ELECTRON MICROSCOPYf_dihedral_angle_d16.6018928
ELECTRON MICROSCOPYf_chiral_restr0.0493508
ELECTRON MICROSCOPYf_plane_restr0.0073927

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