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- PDB-8fyc: Cryo-EM structure of Cas1:Cas2-DEDDh:half-site integration comple... -

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Basic information

Entry
Database: PDB / ID: 8fyc
TitleCryo-EM structure of Cas1:Cas2-DEDDh:half-site integration complex linear CRISPR repeat conformation
Components
  • Cas1
  • Cas2-DEDDh
  • DEDDh
  • DNA (14-MER)
  • DNA (31-MER)
  • DNA (57-MER)
  • DNA/RNA (43-MER)
KeywordsDNA BINDING PROTEIN/DNA / CRISPR / integrase / CRISPR adaptation module / PAM / prespacer / exonuclease / DNA binding protein-DNA complex / enzyme / ribonucleoprotein
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesMegasphaera (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsSkopintsev, P. / Tuck, O.T. / Soczek, K.M. / Doudna, J.
Funding support United States, Switzerland, 3items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DGE 1752814 United States
National Science Foundation (NSF, United States)DGE 2146752 United States
Swiss National Science FoundationP2EZP3_195621 Switzerland
CitationJournal: Nature / Year: 2023
Title: Genome expansion by a CRISPR trimmer-integrase.
Authors: Joy Y Wang / Owen T Tuck / Petr Skopintsev / Katarzyna M Soczek / Gary Li / Basem Al-Shayeb / Julia Zhou / Jennifer A Doudna /
Abstract: CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems ...CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR-Cas systems lack Cas4. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.
History
DepositionJan 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 14, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year / _citation_author.name
Revision 1.2Jun 28, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jul 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
K: DEDDh
J: DNA/RNA (43-MER)
D: Cas2-DEDDh
A: Cas2-DEDDh
B: Cas1
C: Cas1
E: Cas1
F: Cas1
G: DNA (57-MER)
H: DNA (31-MER)
I: DNA (14-MER)


Theoretical massNumber of molelcules
Total (without water)242,81911
Polymers242,81911
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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Protein , 3 types, 7 molecules KDABCEF

#1: Protein DEDDh


Mass: 18652.561 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Megasphaera (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein Cas2-DEDDh


Mass: 10661.198 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Megasphaera (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta
#4: Protein
Cas1


Mass: 34534.512 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Megasphaera (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta

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DNA chain , 4 types, 4 molecules JGHI

#2: DNA chain DNA/RNA (43-MER)


Mass: 23953.268 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Megasphaera (bacteria)
#5: DNA chain DNA (57-MER)


Mass: 17569.213 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Megasphaera (bacteria)
#6: DNA chain DNA (31-MER)


Mass: 9528.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Megasphaera (bacteria)
#7: DNA chain DNA (14-MER)


Mass: 13655.844 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Megasphaera (bacteria)

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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: Cas1:Cas2-DEDDh:half-site integration complex / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.272 MDa / Experimental value: NO
Source (natural)Organism: Megasphaera (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEM3.8.7image acquisition
4cryoSPARC4.1.1CTF correctionPatchCTF
9PHENIX1.19.2-4158model refinement
10cryoSPARC4.1.1initial Euler assignment
11cryoSPARC4.1.1final Euler assignment
12cryoSPARC4.1.1classification
13cryoSPARC4.1.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58475 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00415146
ELECTRON MICROSCOPYf_angle_d0.68321110
ELECTRON MICROSCOPYf_dihedral_angle_d20.5825858
ELECTRON MICROSCOPYf_chiral_restr0.0462379
ELECTRON MICROSCOPYf_plane_restr0.0042203

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