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- EMDB-27160: Type I-C Cas4-Cas1-Cas2 complex bound to a PAM/Processed prespacer -
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Open data
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Basic information
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Title | Type I-C Cas4-Cas1-Cas2 complex bound to a PAM/Processed prespacer | |||||||||
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Function / homology | ![]() 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / iron-sulfur cluster binding / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / magnesium ion binding / protein homodimerization activity ...5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / iron-sulfur cluster binding / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / magnesium ion binding / protein homodimerization activity / DNA binding / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.41 Å | |||||||||
![]() | Dhingra Y / Suresh SK / Juneja P / Sashital DG | |||||||||
Funding support | ![]()
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![]() | ![]() Title: PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation. Authors: Yukti Dhingra / Shravanti K Suresh / Puneet Juneja / Dipali G Sashital / ![]() Abstract: Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of ...Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 128.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20 KB 20 KB | Display Display | ![]() |
Images | ![]() | 77.8 KB | ||
Others | ![]() ![]() | 200.6 MB 200.6 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8d3mMC ![]() 8d3lC ![]() 8d3pC ![]() 8d3qC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 0.9063 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_27160_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27160_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
Entire | Name: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate |
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Components |
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-Supramolecule #1: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
Supramolecule | Name: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: CRISPR-associated endonuclease Cas1
Macromolecule | Name: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125 |
Molecular weight | Theoretical: 39.39334 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITFL TKNGRFLARV VGESRGNVV LRKTQYRISE NDQESTKIAR NFITGKVYNS KWMLERMTRE HPLRVNVEQF KATSQLLSVM MQEIRNCDSL E SLRGWEGQ ...String: MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITFL TKNGRFLARV VGESRGNVV LRKTQYRISE NDQESTKIAR NFITGKVYNS KWMLERMTRE HPLRVNVEQF KATSQLLSVM MQEIRNCDSL E SLRGWEGQ AAINYNKVFD QMILQQKEEF AFHGRSRRPP KDNVNAMLSF AYTLLANDVA AALETVGLDA YVGFMHQDRP GR ASLALDL MEELRGLYAD RFVLSLINRK EMTADGFYKK ENGAVLMTDE ARKTFLKAWQ TKKQEKITHP YLGEKMSWGL VPY VQALLL ARFLRGDLDE YPPFLWK |
-Macromolecule #2: CRISPR-associated endonuclease Cas2
Macromolecule | Name: CRISPR-associated endonuclease Cas2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125 |
Molecular weight | Theoretical: 11.038668 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: GSMLVLITYD VQTSSMGGTK RLRKVAKACQ NYGQRVQNSV FECIVDSTQL TSLKLELTSL IDEEKDSLRI YRLGNNYKTK VEHIGAKPS IDLEDPLIF |
-Macromolecule #5: CRISPR-associated exonuclease Cas4
Macromolecule | Name: CRISPR-associated exonuclease Cas4 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO EC number: 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 25.403396 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: ASNEEDRYLM LSGLQHFQFC KRQWALIHIE QQWEENVRTI EGQHLHKKAD QPFMKEKRGS KLTVRAMPIQ SKNLQISGIC DVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA L RDKVVQMA ...String: ASNEEDRYLM LSGLQHFQFC KRQWALIHIE QQWEENVRTI EGQHLHKKAD QPFMKEKRGS KLTVRAMPIQ SKNLQISGIC DVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA L RDKVVQMA KEMHHYYENR HTPKVKTGPF CNNCSLQSIC LPKLMNKRSV KRYIEGRLSE |
-Macromolecule #3: PAM/processed strand 2
Macromolecule | Name: PAM/processed strand 2 / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 10.139498 KDa |
Sequence | String: (DG)(DT)(DT)(DC)(DT)(DG)(DG)(DT)(DG)(DG) (DT)(DC)(DC)(DT)(DC)(DA)(DG)(DC)(DT)(DA) (DC)(DG)(DT)(DT)(DT)(DT)(DT)(DT)(DG) (DA)(DA)(DT)(DT) |
-Macromolecule #4: PAM/Processed strand 1
Macromolecule | Name: PAM/Processed strand 1 / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 9.840351 KDa |
Sequence | String: (DC)(DG)(DT)(DA)(DG)(DC)(DT)(DG)(DA)(DG) (DG)(DA)(DC)(DC)(DA)(DC)(DC)(DA)(DG)(DA) (DA)(DC)(DT)(DT)(DT)(DT)(DT)(DT)(DG) (DA)(DA)(DT) |
-Macromolecule #6: MANGANESE (II) ION
Macromolecule | Name: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MN |
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Molecular weight | Theoretical: 54.938 Da |
-Macromolecule #7: IRON/SULFUR CLUSTER
Macromolecule | Name: IRON/SULFUR CLUSTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: SF4 |
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Molecular weight | Theoretical: 351.64 Da |
Chemical component information | ![]() ChemComp-FS1: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.75 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol, 2 mM DTT, and 2 mM MnCl2 |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | TFS GLACIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
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Image processing
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 228000 |
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Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |