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- PDB-8flj: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and ... -

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Basic information

Entry
Database: PDB / ID: 8flj
TitleCas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNA
Components
  • (CRISPR-associated ...) x 2
  • (Integration host factor subunit ...Lambda phage) x 2
  • CRISPR leader and repeat, anti-sense strand of DNA
  • CRISPR leader, sense strand of DNA
  • CRISPR repeat and prespacer, sense strand of DNA
  • Prespacer, anti-sense strand of DNA
KeywordsRECOMBINATION / DNA BINDING PROTEIN / HYDROLASE/DNA / 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 / HYDROLASE-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / DNA endonuclease activity / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of chromatin / regulation of translation / chromosome / defense response to virus / DNA recombination / Hydrolases; Acting on ester bonds ...maintenance of CRISPR repeat elements / DNA endonuclease activity / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of chromatin / regulation of translation / chromosome / defense response to virus / DNA recombination / 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
Helicase Cas3, CRISPR-associated, Yersinia-type / : / : / CRISPR-associated nuclease/helicase Cas3, I-F/YPEST, Cas2 domain / CRISPR-associated Cas3 subtype I-F/YPEST-like, C-terminal domain / CRISPR-associated protein Cas1, YPEST subtype / Integration host factor, alpha subunit / Integration host factor, beta subunit / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily ...Helicase Cas3, CRISPR-associated, Yersinia-type / : / : / CRISPR-associated nuclease/helicase Cas3, I-F/YPEST, Cas2 domain / CRISPR-associated Cas3 subtype I-F/YPEST-like, C-terminal domain / CRISPR-associated protein Cas1, YPEST subtype / Integration host factor, alpha subunit / Integration host factor, beta subunit / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / Cas3, HD domain / HD Cas3-type domain profile. / Histone-like DNA-binding protein, conserved site / Bacterial histone-like DNA-binding proteins signature. / Histone-like DNA-binding protein / Bacterial DNA-binding protein / bacterial (prokaryotic) histone like domain / CRISPR-associated endonuclease Cas1, N-terminal domain / Integration host factor (IHF)-like DNA-binding domain superfamily / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / CRISPR-associated endonuclease Cas1 / CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST / Integration host factor subunit alpha / Integration host factor subunit beta
Similarity search - Component
Biological speciesPseudomonas aeruginosa PA14 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.48 Å
AuthorsSantiago-Frangos, A. / Henriques, W.S. / Wiegand, T. / Gauvin, C. / Buyukyoruk, M. / Neselu, K. / Eng, E.T. / Lander, G.C. / Wiedenheft, B.
Funding support United States, 11items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM147842 United States
Life Sciences Research Foundation
Simons Foundation United States
M.J. Murdock Charitable Trust
National Science Foundation (NSF, United States)1828765 United States
NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy programGM129539
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM134867 United States
Amgen
Montana State University Agricultural Experimental Station (USDA NIFA)
VIRIS Detection Systems
CitationJournal: Nat Struct Mol Biol / Year: 2023
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
DepositionDec 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Database references / Category: citation / citation_author / Item: _citation.title / _citation_author.name
Revision 1.2Sep 27, 2023Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Nov 22, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas1
B: CRISPR-associated endonuclease Cas1
C: CRISPR-associated endonuclease Cas1
D: CRISPR-associated endonuclease Cas1
E: Integration host factor subunit alpha
F: Integration host factor subunit beta
G: Integration host factor subunit alpha
H: Integration host factor subunit beta
I: CRISPR leader, sense strand of DNA
J: CRISPR leader and repeat, anti-sense strand of DNA
K: CRISPR repeat and prespacer, sense strand of DNA
L: Prespacer, anti-sense strand of DNA
M: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
N: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST


Theoretical massNumber of molelcules
Total (without water)562,66214
Polymers562,66214
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, The I-F integration complex was assembled from purified DNAs, IHF and Cas1-2/3 as described in the methods section, and the assembled complex was further purified by size- ...Evidence: gel filtration, The I-F integration complex was assembled from purified DNAs, IHF and Cas1-2/3 as described in the methods section, and the assembled complex was further purified by size-exclusion chromatography (SEC) (Superdex 200 10/300, Cytiva). Individual fractions were collected along the elution profile, and were concentrated and stored separately for further analysis and imaging. Individual SEC fractions were analyzed by SDS-PAGE to determine which fractions contained all the proteins necessary for a complete complex. Individual SEC fractions were phenol-chloroform extracted, and the aqueous layer was analyzed by Urea-PAGE to determine which fractions contained all four DNA strands necessary for a complete complex. The fraction chosen for cryo-EM analysis contains all polypeptides and DNA strands required for a complete complex.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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CRISPR-associated ... , 2 types, 6 molecules ABCDMN

#1: Protein
CRISPR-associated endonuclease Cas1


Mass: 38267.148 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PA14 (bacteria) / Gene: cas1, PA14_33350 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q02ML7, Hydrolases; Acting on ester bonds
#8: Protein CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST


Mass: 121273.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PA14 (bacteria) / Gene: cas3, PA14_33340 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q02ML8, Hydrolases; Acting on ester bonds, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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Integration host factor subunit ... , 2 types, 4 molecules EGFH

#2: Protein Integration host factor subunit alpha / Lambda phage / IHF-alpha


Mass: 11646.299 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PA14 (bacteria) / Gene: ihfA, himA, PA14_28720 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02NN5
#3: Protein Integration host factor subunit beta / Lambda phage / IHF-beta


Mass: 10805.378 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PA14 (bacteria) / Gene: ihfB, himD, PA14_23340 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02PW7

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DNA chain , 4 types, 4 molecules IJKL

#4: DNA chain CRISPR leader, sense strand of DNA


Mass: 42935.559 Da / Num. of mol.: 1
Mutation: C30A,T31A,T32A,C34A,G35A,G97A,G98A,T99A,T101A,T102A,T103C,C104G,T105C,T108C,T109G,C110A,C111A,T112A,A117C,T118G
Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa PA14 (bacteria)
#5: DNA chain CRISPR leader and repeat, anti-sense strand of DNA


Mass: 52701.645 Da / Num. of mol.: 1
Mutation: A54G,T55G,A60T,G61T,G62T,A63C,A64G,A67G,G68C,A69G,A70T,A71T,A73T,C74T,C75T,C137T,G138T,A140T,A141T,G142T
Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa PA14 (bacteria)
#6: DNA chain CRISPR repeat and prespacer, sense strand of DNA


Mass: 18241.707 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain Prespacer, anti-sense strand of DNA


Mass: 8263.364 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNACOMPLEXall0MULTIPLE SOURCES
2Cas1-Cas2/3 heterohexameric integraseCOMPLEX#1, #81RECOMBINANT
3IHF heterodimerCOMPLEX#2-#31RECOMBINANT
4CRISPR leader, repeat, and prespacer DNA annealed to mimic a half-site integration intermediate.COMPLEX#4-#71MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11.56 MDaNO
21.395 MDaNO
31NO
41.122 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pseudomonas aeruginosa PA14 (bacteria)652611
33Pseudomonas aeruginosa PA14 (bacteria)652611
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMTris-HClTris1
2200 mMMonopotassium glutamate1
35 mMEDTAEthylenediaminetetraacetic acid1
41 mMTCEP1
50.2 %Glycerol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were glow discharged at 15 mA for 15 seconds with a 10 second hold (easiGlow, Pelco).
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated magnification: 46860 X / Nominal defocus max: 2100 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 69 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 10740
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 11520 / Height: 8184

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refinedev_4674refinement
PHENIXdev_4674refinement
EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
Image processingDetails: Patch motion correction and patch CTF correction were performed in cryoSPARC.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5846923 / Details: Template picking
3D reconstructionResolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 366794 / Num. of class averages: 1 / Symmetry type: POINT
RefinementHighest resolution: 3.48 Å / Cross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005233098
ELECTRON MICROSCOPYf_angle_d0.834846528
ELECTRON MICROSCOPYf_chiral_restr0.05525329
ELECTRON MICROSCOPYf_plane_restr0.01074909
ELECTRON MICROSCOPYf_dihedral_angle_d24.18967290

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