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- PDB-5u0a: CRISPR RNA-guided surveillance complex -

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Basic information

Entry
Database: PDB / ID: 5u0a
TitleCRISPR RNA-guided surveillance complex
Components
  • (CRISPR-associated protein, ...) x 4
  • Cse2Carbon diselenide
  • Nontarget Strand
  • Target Strand
  • crRNA
KeywordsIMMUNE SYSTEM / CRISPR-Cas / Cascacde / surveillance
Function / homologyCse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse3 / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse2 / CRISPR-associated protein Cas5, N-terminal / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) ...Cse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse3 / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse2 / CRISPR-associated protein Cas5, N-terminal / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR associated protein / CT1975-like protein / CRISPR-associated protein Cse1 (CRISPR_cse1) / maintenance of CRISPR repeat elements / defense response to virus / RNA binding / identical protein binding / CRISPR-associated protein, Cse1 family / Uncharacterized protein / CRISPR-associated protein, Cse4 family / CRISPR-associated protein, Cas5e family / CRISPR-associated protein, Cse3 family
Function and homology information
Specimen sourceThermobifida fusca (bacteria)
Thermobifida fusca YX (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.3 Å resolution
AuthorsXiao, Y. / Luo, M. / Hayes, R.P. / Kim, J. / Ng, S. / Ding, F. / Liao, M. / Ke, A.
CitationJournal: Cell / Year: 2017
Title: Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.
Authors: Yibei Xiao / Min Luo / Robert P Hayes / Jonathan Kim / Sherwin Ng / Fang Ding / Maofu Liao / Ailong Ke
Abstract: Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two ...Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 23, 2016 / Release: Aug 9, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Aug 9, 2017Structure modelrepositoryInitial release
1.1Aug 16, 2017Structure modelDatabase referencescitation_citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: CRISPR-associated protein, Cse3 family
C: CRISPR-associated protein, Cse1 family
D: CRISPR-associated protein, Cse4 family
E: CRISPR-associated protein, Cse4 family
F: CRISPR-associated protein, Cse4 family
G: CRISPR-associated protein, Cse4 family
H: CRISPR-associated protein, Cse4 family
I: CRISPR-associated protein, Cse4 family
J: Cse2
K: crRNA
L: Cse2
M: Target Strand
N: CRISPR-associated protein, Cas5e family
O: Nontarget Strand


Theoretical massNumber of molelcules
Total (without water)464,31114
Polyers464,31114
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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CRISPR-associated protein, ... , 4 types, 9 molecules ACDEFGHIN

#1: Protein/peptide CRISPR-associated protein, Cse3 family


Mass: 26327.938 Da / Num. of mol.: 1
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1588 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ5
#2: Protein/peptide CRISPR-associated protein, Cse1 family


Mass: 61433.297 Da / Num. of mol.: 1
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1592 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ1
#3: Protein/peptide
CRISPR-associated protein, Cse4 family / Cas7


Mass: 41043.043 Da / Num. of mol.: 6
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1590 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ3
#7: Protein/peptide CRISPR-associated protein, Cas5e family


Mass: 28279.260 Da / Num. of mol.: 1
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1589 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ4

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DNA chain , 2 types, 2 molecules MO

#6: DNA chain Target Strand


Mass: 15251.741 Da / Num. of mol.: 1 / Source: (synth.) Thermobifida fusca YX (bacteria)
#8: DNA chain Nontarget Strand


Mass: 12076.723 Da / Num. of mol.: 1 / Source: (synth.) Thermobifida fusca YX (bacteria)

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Protein/peptide / RNA chain , 2 types, 3 molecules JLK

#4: Protein/peptide Cse2 / Carbon diselenide


Mass: 27446.613 Da / Num. of mol.: 2
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1591 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q47PJ2
#5: RNA chain crRNA


Mass: 19790.793 Da / Num. of mol.: 1 / Source: (synth.) Thermobifida fusca YX (bacteria) / Details: RNA was prepared by in vitro transcription

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: CRISPR RNA-guided surveillance complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: RECOMBINANT
Source (natural)Organism: Thermobifida fusca (strain YX) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pcdf
Buffer solutionDetails: 10 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT / pH: 7.5
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: 400 mesh Quantifoil holey carbon grid
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 85 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 / Calibrated defocus min: 1000 nm / Calibrated defocus max: 21000 nm / Cs: 2 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: GATAN LIQUID NITROGEN / Temperature (max): 105 kelvins / Temperature (min): 80 kelvins
Image recordingElectron dose: 8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of real images: 1072

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Processing

SoftwareName: PHENIX / Version: 1.11.1-2575_1692: / Classification: refinement
EM software
IDNameVersionCategory
1Spiderparticle selection
2SAMUELparticle selection
3UcsfImager4image acquisition
5RELION1.4CTF correction
8COOT0.8.1model fitting
10SPIDERinitial Euler assignment
11RELION1.4final Euler assignment
12RELION1.4classification
13RELION1.43D reconstruction
14PHENIX1.11.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 303301
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 131292 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01330167
ELECTRON MICROSCOPYf_angle_d1.42041600
ELECTRON MICROSCOPYf_dihedral_angle_d18.35417710
ELECTRON MICROSCOPYf_chiral_restr0.0794700
ELECTRON MICROSCOPYf_plane_restr0.0084928

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