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- PDB-6c66: CRISPR RNA-guided surveillance complex, pre-nicking -

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Basic information

Entry
Database: PDB / ID: 6c66
TitleCRISPR RNA-guided surveillance complex, pre-nicking
Components
  • (CRISPR-associated protein, ...) x 4
  • CRISPR-associated helicase, Cas3 family
  • Nontarget strand
  • Target strand
  • Uncharacterized protein
  • crRNA
KeywordsDNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / Cascade / Cas3 / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


nuclease activity / maintenance of CRISPR repeat elements / defense response to virus / RNA helicase activity / RNA binding / ATP binding / metal ion binding / identical protein binding
Similarity search - Function
Cas3 C-terminal domain / Hypothetical protein af1432 - #30 / : / Helicase Cas3, CRISPR-associated, core / Cas3, HD domain / CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse2 / Cse2 superfamily / CT1975-like protein / CRISPR-associated protein Cse2 (CRISPR_cse2) ...Cas3 C-terminal domain / Hypothetical protein af1432 - #30 / : / Helicase Cas3, CRISPR-associated, core / Cas3, HD domain / CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse2 / Cse2 superfamily / CT1975-like protein / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR associated protein / CRISPR_assoc / : / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / CRISPR-associated nuclease/helicase Cas3, C-terminal / HD Cas3-type domain profile. / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / AAA domain / CRISPR-associated protein Cas5, N-terminal / Hypothetical protein af1432 / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Helicase, C-terminal / P-loop containing nucleoside triphosphate hydrolase / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated helicase, Cas3 family / CRISPR-associated protein, Cse1 family / CRISPR-associated protein, Cse2 family / CRISPR-associated protein, Cse4 family / CRISPR-associated protein, Cas5e family / CRISPR-associated protein, Cse3 family
Similarity search - Component
Biological speciesThermobifida fusca (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsXiao, Y. / Luo, M. / Liao, M. / Ke, A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118174 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM102543 United States
CitationJournal: Science / Year: 2018
Title: Structure basis for RNA-guided DNA degradation by Cascade and Cas3.
Authors: Yibei Xiao / Min Luo / Adam E Dolan / Maofu Liao / Ailong Ke /
Abstract: Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and ...Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo-electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop-forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5'-triphosphate-dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems.
History
DepositionJan 17, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 27, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Feb 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization
Revision 1.4Nov 29, 2023Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_related / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_related.db_name / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
G: CRISPR-associated helicase, Cas3 family
A: CRISPR-associated protein, Cse1 family
B: CRISPR-associated protein, Cse4 family
C: CRISPR-associated protein, Cse4 family
D: CRISPR-associated protein, Cse4 family
E: CRISPR-associated protein, Cse4 family
F: CRISPR-associated protein, Cse4 family
H: CRISPR-associated protein, Cse4 family
I: Uncharacterized protein
J: crRNA
K: Uncharacterized protein
L: Target strand
M: CRISPR-associated protein, Cas5e family
N: Nontarget strand
O: CRISPR-associated protein, Cse3 family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)575,03517
Polymers574,92415
Non-polymers1122
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 3 molecules GIK

#1: Protein CRISPR-associated helicase, Cas3 family


Mass: 104039.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1593 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ0
#4: Protein Uncharacterized protein


Mass: 27446.613 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1591 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ2

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

#2: Protein CRISPR-associated protein, Cse1 family


Mass: 61433.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1592 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ1
#3: Protein
CRISPR-associated protein, Cse4 family


Mass: 41043.043 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1590 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ3
#7: Protein CRISPR-associated protein, Cas5e family


Mass: 28279.260 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1589 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ4
#9: Protein CRISPR-associated protein, Cse3 family


Mass: 26327.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1588 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ5

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DNA chain , 2 types, 2 molecules LN

#6: DNA chain Target strand


Mass: 16801.725 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermobifida fusca (bacteria)
#8: DNA chain Nontarget strand


Mass: 17099.920 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermobifida fusca (bacteria)

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RNA chain / Non-polymers , 2 types, 3 molecules J

#10: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe
#5: RNA chain crRNA


Mass: 19790.793 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (bacteria) / Production host: Escherichia coli (E. coli)

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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: CRISPR RNA-guided surveillance complex with Cas3 bound in its pre-nicking state
Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Source (natural)Organism: Thermobifida fusca (strain YX) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pcdf
Buffer solutionpH: 7.5 / Details: 10 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
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
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 31000 X / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2700 nm / Cs: 2 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: GATAN LIQUID NITROGEN / Temperature (max): 105 K / Temperature (min): 80 K
Image recordingElectron dose: 8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1428

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1SPIDERparticle selection
2RELIONparticle selection
3UCSFImage44image acquisition
5RELION1.4CTF correction
8Coot0.8.1model fitting
10PHENIX1.11.1model refinement
11SPIDERinitial Euler assignment
12RELION1.4final Euler assignment
13RELION1.4classification
14RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 322268
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51889 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00837324
ELECTRON MICROSCOPYf_angle_d1.09551376
ELECTRON MICROSCOPYf_dihedral_angle_d14.47721993
ELECTRON MICROSCOPYf_chiral_restr0.0575814
ELECTRON MICROSCOPYf_plane_restr0.0076200

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