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- EMDB-29563: Cryo-EM structure of Cas1:Cas2-DEDDh:half-site integration complex -

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Basic information

Entry
Database: EMDB / ID: EMD-29563
TitleCryo-EM structure of Cas1:Cas2-DEDDh:half-site integration complex
Map data
Sample
  • Complex: Cas1:Cas2-DEDDh:half-site integration complex
    • Protein or peptide: Cas2-DEDDh
    • Protein or peptide: Cas1
    • DNA: DNA (52-MER)
    • DNA: DNA (31-MER)
    • DNA: DNA (36-MER)
    • DNA: DNA (17-MER)
KeywordsCRISPR / integrase / CRISPR adaptation module / PAM / prespacer / exonuclease / DNA binding protein-DNA complex / enzyme / ribonucleoprotein
Biological speciesMegasphaera (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSkopintsev P / Tuck OT / Soczek KM / Doudna J
Funding support United States, Switzerland, 3 items
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, 2023-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29563.png

Downloads & links

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Map

FileDownload / File: emd_29563.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.115 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-1.0680313 - 1.4651513
Average (Standard dev.)-0.0006929422 (±0.030866072)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 267.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29563_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_29563_additional_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29563_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29563_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cas1:Cas2-DEDDh:half-site integration complex

EntireName: Cas1:Cas2-DEDDh:half-site integration complex
Components
  • Complex: Cas1:Cas2-DEDDh:half-site integration complex
    • Protein or peptide: Cas2-DEDDh
    • Protein or peptide: Cas1
    • DNA: DNA (52-MER)
    • DNA: DNA (31-MER)
    • DNA: DNA (36-MER)
    • DNA: DNA (17-MER)

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Supramolecule #1: Cas1:Cas2-DEDDh:half-site integration complex

SupramoleculeName: Cas1:Cas2-DEDDh:half-site integration complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Megasphaera (bacteria)
Molecular weightTheoretical: 272 KDa

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Macromolecule #1: Cas2-DEDDh

MacromoleculeName: Cas2-DEDDh / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Megasphaera (bacteria)
Molecular weightTheoretical: 32.632615 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPMTVITLKN VPQSLRGDLT RWMQEIATGV YVGNFNSRIR EYLWRRVQET MGAGEASMCF AARNELGYDF LTENASRSVI DYDGLPLIF IPKEQSAVSD LPKGFSTAAK LHRAHIAGSG KKKEKPIRYV VIDIETDGKD AKRNHILEIG AIRCEDGKET H FTALISGD ...String:
MPMTVITLKN VPQSLRGDLT RWMQEIATGV YVGNFNSRIR EYLWRRVQET MGAGEASMCF AARNELGYDF LTENASRSVI DYDGLPLIF IPKEQSAVSD LPKGFSTAAK LHRAHIAGSG KKKEKPIRYV VIDIETDGKD AKRNHILEIG AIRCEDGKET H FTALISGD AVPPSITKLT GITATLLQKE GQEEKKVLTA FREFIGDDDL VGYHVSFDIE FLRQAFKKYG LGYLKNKTHD LL RIVKKEQ LFQADYKLET SLQSYGIHKK VPHRALGDAE LVKCLAKKLN KF

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Macromolecule #2: Cas1

MacromoleculeName: Cas1 / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Megasphaera (bacteria)
Molecular weightTheoretical: 35.022074 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGPIIAGKS ESSELPRVED RATFIYIEHA KINRVDSAVT VAEAKGVVRI PAAMIGVLLL GPGTDISHRA VELLGDTGTA LVWVGEQGV RYYASGRALA RSTRFLVKQA ELVTNERSRL RVARRMYQMR FPTEDVSKLT MQQLRSHEGA RVRRKYRELS K KYNVPWKK ...String:
MAGPIIAGKS ESSELPRVED RATFIYIEHA KINRVDSAVT VAEAKGVVRI PAAMIGVLLL GPGTDISHRA VELLGDTGTA LVWVGEQGV RYYASGRALA RSTRFLVKQA ELVTNERSRL RVARRMYQMR FPTEDVSKLT MQQLRSHEGA RVRRKYRELS K KYNVPWKK RVYNPDDFAG GDPINQALSA AHVALYGLVH SVVAALGLSP GLGFVHTGHD RSFIYDVADL YKAEITVPIA FA VAAEAEE GQDIGQLARL RTRDAFVDGK ILKRMVKDLQ TLLEIPEEGQ IEAEPLSLWD DKEKLVPYGV NYSEVTSCP

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Macromolecule #3: DNA (52-MER)

MacromoleculeName: DNA (52-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Megasphaera (bacteria)
Molecular weightTheoretical: 19.721586 KDa
SequenceString: (DA)(DG)(DA)(DT)(DT)(DG)(DA)(DG)(DA)(DC) (DC)(DA)(DG)(DG)(DT)(DC)(DT)(DC)(DC)(DG) (DT)(DT)(DT)(DC)(DA)(DT)(DG)(DA)(DG) (DT)(DC)(DT)(DT)(DT)(DC)(DC)(DC)(DG)(DC) (DA) (DC)(DG)(DA)(DG)(DC)(DG) ...String:
(DA)(DG)(DA)(DT)(DT)(DG)(DA)(DG)(DA)(DC) (DC)(DA)(DG)(DG)(DT)(DC)(DT)(DC)(DC)(DG) (DT)(DT)(DT)(DC)(DA)(DT)(DG)(DA)(DG) (DT)(DC)(DT)(DT)(DT)(DC)(DC)(DC)(DG)(DC) (DA) (DC)(DG)(DA)(DG)(DC)(DG)(DG)(DG) (DG)(DG)(DT)(DG)(DA)(DT)(DC)(DC)(DC)(DA) (DC)(DG) (DC)(DG)(DC)(DA)

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Macromolecule #4: DNA (31-MER)

MacromoleculeName: DNA (31-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Megasphaera (bacteria)
Molecular weightTheoretical: 10.106497 KDa
SequenceString:
(DA)(DA)(DA)(DC)(DG)(DG)(DA)(DG)(DA)(DC) (DC)(DT)(DG)(DG)(DT)(DC)(DT)(DC)(DA)(DA) (DT)(DC)(DT)(DG)(DC)(DG)(DT)(DG)(DT) (DT)(DC)(DC)(DC)

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Macromolecule #5: DNA (36-MER)

MacromoleculeName: DNA (36-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Megasphaera (bacteria)
Molecular weightTheoretical: 23.953268 KDa
SequenceString: (DT)(DG)(DC)(DG)(DC)(DG)(DT)(DG)(DG)(DG) (DA)(DT)(DC)(DA)(DC)(DC)(DC)(DC)(DC)(DG) (DC)(DT)(DC)(DG)(DT)(DG)(DC)(DG)(DG) (DG)(DA)(DA)(DA)(DG)(DA)(DC)(DA)(DG)(DT) (DA) (DA)(DT)(DG)(DG)(DA)(DT) ...String:
(DT)(DG)(DC)(DG)(DC)(DG)(DT)(DG)(DG)(DG) (DA)(DT)(DC)(DA)(DC)(DC)(DC)(DC)(DC)(DG) (DC)(DT)(DC)(DG)(DT)(DG)(DC)(DG)(DG) (DG)(DA)(DA)(DA)(DG)(DA)(DC)(DA)(DG)(DT) (DA) (DA)(DT)(DG)(DG)(DA)(DT)(DT)(DC) (DC)(DT)(DT)(DT)(DA)(DT)(DT)(DT)(DT)(DC) (DG)(DC) (DC)(DC)(DT)(DT)(DT)(DT)(DA) (DC)(DG)(DC)(DT)(DT)(DA)(DC)(DT)(DG)(DA) (DC)

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Macromolecule #6: DNA (17-MER)

MacromoleculeName: DNA (17-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Megasphaera (bacteria)
Molecular weightTheoretical: 13.655844 KDa
SequenceString:
(DA)(DC)(DG)(DT)(DC)(DA)(DG)(DT)(DA)(DA) (DG)(DC)(DG)(DT)(DA)(DA)(DA)(DA)(DG)(DG) (DG)(DC)(DG)(DA)(DA)(DA)(DA)(DT)(DA) (DA)(DA)(DG)(DG)(DA)(DA)(DT)(DC)(DC)(DA) (DT) (DT)(DA)(DC)(DT)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab-initio map calculated in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.1.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.1) / Number images used: 1048353
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8fyb:
Cryo-EM structure of Cas1:Cas2-DEDDh:half-site integration complex

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