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- EMDB-29280: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and ... -
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Open data
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Basic information
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Title | Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNA | ||||||||||||||||||||||||||||||||||||
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![]() | DNA integration / maintenance of CRISPR repeat elements / IHF-DNA complex / Enzymes altering nucleic acid conformation / Site specific endodeoxyribonucleases: cleavage is not sequence specific / Nucleotidyltransferases / GO:0008301 / RECOMBINATION / DNA BINDING PROTEIN / HYDROLASE-DNA complex | ||||||||||||||||||||||||||||||||||||
Function / homology | ![]() maintenance of CRISPR repeat elements / helicase activity / DNA endonuclease activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of chromatin / regulation of translation / chromosome / DNA recombination / defense response to virus / Hydrolases; Acting on ester bonds ...maintenance of CRISPR repeat elements / helicase activity / DNA endonuclease activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of chromatin / regulation of translation / chromosome / DNA recombination / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / regulation of DNA-templated transcription / DNA binding / ATP binding / identical protein binding / metal ion binding Similarity search - Function | ||||||||||||||||||||||||||||||||||||
Biological species | ![]() | ||||||||||||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.48 Å | ||||||||||||||||||||||||||||||||||||
![]() | Santiago-Frangos A / Henriques WS / Wiegand T / Gauvin C / Buyukyoruk M / Neselu K / Eng ET / Lander GC / Wiedenheft B | ||||||||||||||||||||||||||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays. Authors: Andrew Santiago-Frangos / William S Henriques / Tanner Wiegand / Colin C Gauvin / Murat Buyukyoruk / Ava B Graham / Royce A Wilkinson / Lenny Triem / Kasahun Neselu / Edward T Eng / Gabriel ...Authors: Andrew Santiago-Frangos / William S Henriques / Tanner Wiegand / Colin C Gauvin / Murat Buyukyoruk / Ava B Graham / Royce A Wilkinson / Lenny Triem / Kasahun Neselu / Edward T Eng / Gabriel C Lander / Blake Wiedenheft / ![]() Abstract: Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration ...Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560-kDa integration complex structure that explains how Pseudomonas aeruginosa Cas (Cas1-Cas2/3) and non-Cas proteins (for example, integration host factor) fold 150 base pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3 proteins rotate 100 degrees to expose DNA-binding sites on either side of the Cas2 homodimer, which each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5'-GT. Collectively, this work reveals new DNA-binding surfaces on Cas2 that are critical for DNA folding and site-specific delivery of foreign DNA. | ||||||||||||||||||||||||||||||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 202.6 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 32 KB 32 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 14.4 KB | Display | ![]() |
Images | ![]() | 68 KB | ||
Filedesc metadata | ![]() | 9 KB | ||
Others | ![]() ![]() | 200.3 MB 200.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 864 KB | Display | ![]() |
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Full document | ![]() | 863.5 KB | Display | |
Data in XML | ![]() | 22 KB | Display | |
Data in CIF | ![]() | 28.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8fljMC M: atomic model generated by this map C: citing same article ( |
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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: 1.067 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_29280_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_29280_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
+Entire : Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and ...
+Supramolecule #1: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and ...
+Supramolecule #2: Cas1-Cas2/3 heterohexameric integrase
+Supramolecule #3: IHF heterodimer
+Supramolecule #4: CRISPR leader, repeat, and prespacer DNA annealed to mimic a half...
+Macromolecule #1: CRISPR-associated endonuclease Cas1
+Macromolecule #2: Integration host factor subunit alpha
+Macromolecule #3: Integration host factor subunit beta
+Macromolecule #8: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
+Macromolecule #4: CRISPR leader, sense strand of DNA
+Macromolecule #5: CRISPR leader and repeat, anti-sense strand of DNA
+Macromolecule #6: CRISPR repeat and prespacer, sense strand of DNA
+Macromolecule #7: Prespacer, anti-sense strand of DNA
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. Details: Grids were glow discharged at 15 mA for 15 seconds with a 10 second hold (easiGlow, Pelco). | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number real images: 10740 / Average exposure time: 2.5 sec. / Average electron dose: 69.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 46860 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 81000 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |