EMDB-29280: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and ...

Basic information

Entry

Database: EMDB / ID: EMD-29280

• Title: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNA

Sample

• Complex: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNA
  • Complex: Cas1-Cas2/3 heterohexameric integrase
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
  • Complex: IHF heterodimer
    • Protein or peptide: Integration host factor subunit alphaLambda phage
    • Protein or peptide: Integration host factor subunit betaLambda phage
  • Complex: CRISPR leader, repeat, and prespacer DNA annealed to mimic a half-site integration intermediate.
    • DNA: CRISPR leader, sense strand of DNA
    • DNA: CRISPR leader and repeat, anti-sense strand of DNA
    • DNA: CRISPR repeat and prespacer, sense strand of DNA
    • DNA: Prespacer, anti-sense strand of DNA

• Keywords: 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

Function:

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

Domain/homology:

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

Component:

CRISPR-associated endonuclease Cas1 / CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST / Integration host factor subunit alpha / Integration host factor subunit beta

• Biological species: Pseudomonas aeruginosa PA14 (bacteria) / synthetic construct (others)
• Method: single particle reconstruction / cryo EM / Resolution: 3.48 Å
• Authors: Santiago-Frangos A / Henriques WS / Wiegand T / Gauvin C / Buyukyoruk M / Neselu K / Eng ET / Lander GC / Wiedenheft B

Funding support

United States, 11 items

Organization	Grant number	Country
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 program	GM129539
Simons Foundation	SF349247	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

• Citation: Journal: 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 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 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

Deposition	Dec 21, 2022	-
Header (metadata) release	Sep 6, 2023	-
Map release	Sep 6, 2023	-
Update	Nov 22, 2023	-
Current status	Nov 22, 2023	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_29280.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.067 Å

Density

Contour Level	By AUTHOR: 4.6
Minimum - Maximum	-22.553097000000001 - 42.120663
Average (Standard dev.)	0.0007603867 (±1.0002414)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 384 384 384 Spacing 384 384 384
Cell	A=B=C: 409.72803 Å α=β=γ: 90.0 °

Supplemental data

Half map: #2

• File: emd_29280_half_map_1.map

Half map: #1

• File: emd_29280_half_map_2.map

Sample components

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

• Entire: Name: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNA

Components

• Complex: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNA
  • Complex: Cas1-Cas2/3 heterohexameric integrase
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
  • Complex: IHF heterodimer
    • Protein or peptide: Integration host factor subunit alphaLambda phage
    • Protein or peptide: Integration host factor subunit betaLambda phage
  • Complex: CRISPR leader, repeat, and prespacer DNA annealed to mimic a half-site integration intermediate.
    • DNA: CRISPR leader, sense strand of DNA
    • DNA: CRISPR leader and repeat, anti-sense strand of DNA
    • DNA: CRISPR repeat and prespacer, sense strand of DNA
    • DNA: Prespacer, anti-sense strand of DNA

Supramolecule #1: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and ...

• Supramolecule: Name: Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNA; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
• Molecular weight: Theoretical: 122 KDa

Supramolecule #2: Cas1-Cas2/3 heterohexameric integrase

• Supramolecule: Name: Cas1-Cas2/3 heterohexameric integrase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #8
• Source (natural): Organism: Pseudomonas aeruginosa PA14 (bacteria)

Supramolecule #3: IHF heterodimer

• Supramolecule: Name: IHF heterodimer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
• Source (natural): Organism: Pseudomonas aeruginosa PA14 (bacteria)

Supramolecule #4: CRISPR leader, repeat, and prespacer DNA annealed to mimic a half...

• Supramolecule: Name: CRISPR leader, repeat, and prespacer DNA annealed to mimic a half-site integration intermediate.; type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4-#7

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
• Source (natural): Organism: Pseudomonas aeruginosa PA14 (bacteria)
• Molecular weight: Theoretical: 38.267148 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MWSHPQFEKE NLYFQGSMDD ISPSELKTIL HSKRANLYYL QHCRVLVNGG RVEYVTDEGR HSHYWNIPIA NTTSLLLGTG TSITQAAMR ELARAGVLVG FCGGGGTPLF SANEVDVEVS WLTPQSEYRP TEYLQRWVGF WFDEEKRLVA ARHFQRARLE R IRHSWLED RVLRDAGFAV DATALAVAVE DSARALEQAP NHEHLLTEEA RLSKRLFKLA AQATRYGEFV RAKRGSGGDP AN RFLDHGN YLAYGLAATA TWVLGIPHGL AVLHGKTRRG GLVFDVADLI KDSLILPQAF LSAMRGDEEQ DFRQACLDNL SRA QALDFM IDTLKDVAQR STVSA

UniProtKB: CRISPR-associated endonuclease Cas1

Macromolecule #2: Integration host factor subunit alpha

• Macromolecule: Name: Integration host factor subunit alpha / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Pseudomonas aeruginosa PA14 (bacteria)
• Molecular weight: Theoretical: 11.646299 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

GPMGALTKAE IAERLYEELG LNKREAKELV ELFFEEIRQA LEHNEQVKLS GFGNFDLRDK RQRPGRNPKT GEEIPITARR VVTFRPGQK LKARVEAYAG TKS

UniProtKB: Integration host factor subunit alpha

Macromolecule #3: Integration host factor subunit beta

• Macromolecule: Name: Integration host factor subunit beta / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Pseudomonas aeruginosa PA14 (bacteria)
• Molecular weight: Theoretical: 10.805378 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

GPMTKSELIE RIVTHQGQLS AKDVELAIKT MLEQMSQALA TGDRIEIRGF GSFSLHYRAP RVGRNPKTGE SVRLDGKFVP HFKPGKELR DRVNEPE

UniProtKB: Integration host factor subunit beta

Macromolecule #8: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST

• Macromolecule: Name: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST; type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
• Source (natural): Organism: Pseudomonas aeruginosa PA14 (bacteria)
• Molecular weight: Theoretical: 121.27368 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MNILLVSQCE KRALSETRRI LDQFAERRGE RTWQTPITQA GLDTLRRLLK KSARRNTAVA CHWIRGRDHS ELLWIVGDAS RFNAQGAVP TNRTCRDILR KEDENDWHSA EDIRLLTVMA ALFHDIGKAS QAFQAKLRNR GKPMADAYRH EWVSLRLFEA F VGPGSSDE DWLRRLADKR ETGDAWLSQL ARDDRQSAPP GPFQKSRLPP LAQAVGWLIV SHHRLPNGDH RGSASLARLP AP IQSQWCG ARDADAKEKA ACWQFPHGLP FASAHWRART ALCAQSMLER PGLLARGPAL LHDSYVMHVS RLILMLADHH YSS LPADSR LGDPNFPLHA NTDRDSGKLK QRLDEHLLGV ALHSRKLAGT LPRLERQLPR LARHKGFTRR VEQPRFRWQD KAYD CAMAC REQAMEHGFF GLNLASTGCG KTLANGRILY ALADPQRGAR FSIALGLRSL TLQTGQAYRE RLGLGDDDLA ILVGG SAAR ELFEKQQERL ERSGSESAQE LLAENSHVHF AGTLEDGPLR EWLGRNSAGN RLLQAPILAC TIDHLMPASE SLRGGH QIA PLLRLMTSDL VLDEVDDFDI DDLPALSRLV HWAGLFGSRV LLSSATLPPA LVQGLFEAYR SGREIFQRHR GAPGRAT EI RCAWFDEFSS QSSAHGAVTS FSEAHATFVA QRLAKLEQLP PRRQAQLCTV HAAGEARPAL CRELAGQMNT WMADLHRC H HTEHQGRRIS FGLLRLANIE PLIELAQAIL AQGAPEGLHV HLCVYHSRHP LLVRSAIERQ LDELLKRSDD DAAALFARP TLAKALQAST ERDHLFVVLA SPVAEVGRDH DYDWAIVEPS SMRSIIQLAG RIRRHRSGFS GEANLYLLSR NIRSLEGQNP AFQRPGFET PDFPLDSHDL HDLLDPALLA RIDASPRIVE PFPLFPRSRL VDLEHRRLRA LMLADDPPSS LLGVPLWWQT P ASLSGALQ TSQPFRAGAK ERCYALLPDE DDEERLHFSR YEEGTWSNQD NLLRNLDLTY GPRIQTWGTV NYREELVAMA GR EDLDLRQ CAMRYGEVRL RENTQGWSYH PYLGFKKYN

UniProtKB: CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST

Macromolecule #4: CRISPR leader, sense strand of DNA

• Macromolecule: Name: CRISPR leader, sense strand of DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Pseudomonas aeruginosa PA14 (bacteria)
• Molecular weight: Theoretical: 42.935559 KDa

Sequence

String:

(DA)(DA)(DG)(DC)(DT)(DT)(DC)(DC)(DG)(DA) (DC)(DC)(DC)(DT)(DT)(DT)(DT)(DT)(DT)(DC) (DG)(DG)(DA)(DC)(DG)(DA)(DT)(DT)(DT) (DA)(DA)(DA)(DA)(DA)(DA)(DC)(DC)(DC)(DT) (DT) (DA)(DT)(DA)(DA)(DA)(DT)(DC)(DA) (DG)(DC)(DA)(DA)(DG)(DT)(DT)(DA)(DC)(DG) (DA)(DG) (DA)(DC)(DC)(DT)(DC)(DG)(DA) (DA)(DA)(DA)(DA)(DA)(DG)(DA)(DG)(DG)(DG) (DT)(DT)(DT) (DC)(DT)(DG)(DG)(DC)(DG) (DG)(DG)(DA)(DA)(DA)(DA)(DA)(DC)(DT)(DC) (DA)(DA)(DA)(DA) (DA)(DA)(DC)(DG)(DC) (DT)(DT)(DC)(DG)(DA)(DA)(DA)(DT)(DC)(DA) (DA)(DC)(DC)(DG)(DG) (DT)(DT)(DA)(DT) (DA)(DG)(DG)(DT)(DT)(DT)(DT)(DC)(DG)(DG) (DA)(DG)(DC)(DT)(DA)

Macromolecule #5: CRISPR leader and repeat, anti-sense strand of DNA

• Macromolecule: Name: CRISPR leader and repeat, anti-sense strand of DNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Pseudomonas aeruginosa PA14 (bacteria)
• Molecular weight: Theoretical: 52.701645 KDa

Sequence

String:

(DT)(DG)(DA)(DT)(DT)(DT)(DT)(DC)(DT)(DT) (DA)(DG)(DC)(DT)(DG)(DC)(DC)(DT)(DA)(DC) (DA)(DC)(DG)(DG)(DC)(DA)(DG)(DT)(DG) (DA)(DA)(DC)(DT)(DA)(DG)(DC)(DT)(DC)(DC) (DG) (DA)(DA)(DA)(DA)(DC)(DC)(DT)(DA) (DT)(DA)(DA)(DC)(DC)(DG)(DG)(DT)(DT)(DG) (DA)(DT) (DT)(DT)(DC)(DG)(DA)(DA)(DG) (DC)(DG)(DT)(DT)(DT)(DT)(DT)(DT)(DG)(DA) (DG)(DT)(DT) (DT)(DT)(DT)(DC)(DC)(DC) (DG)(DC)(DC)(DA)(DG)(DA)(DA)(DA)(DC)(DC) (DC)(DT)(DC)(DT) (DT)(DT)(DT)(DT)(DT) (DC)(DG)(DA)(DG)(DG)(DT)(DC)(DT)(DC)(DG) (DT)(DA)(DA)(DC)(DT) (DT)(DG)(DC)(DT) (DG)(DA)(DT)(DT)(DT)(DA)(DT)(DA)(DA)(DG) (DG)(DG)(DT)(DT)(DT)(DT) (DT)(DT)(DA) (DA)(DA)(DT)(DC)(DG)(DT)(DC)(DC)(DG)(DA) (DA)(DA)(DA)(DA)(DA)(DG)(DG) (DG)(DT) (DC)(DG)(DG)(DA)(DA)(DG)(DC)(DT)(DT)

Macromolecule #6: CRISPR repeat and prespacer, sense strand of DNA

• Macromolecule: Name: CRISPR repeat and prespacer, sense strand of DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: synthetic construct (others)
• Molecular weight: Theoretical: 18.241707 KDa

Sequence

String:

(DG)(DT)(DG)(DC)(DA)(DA)(DG)(DT)(DC)(DG) (DT)(DT)(DT)(DT)(DG)(DC)(DT)(DA)(DG)(DA) (DG)(DC)(DT)(DA)(DC)(DA)(DT)(DG)(DT) (DT)(DC)(DA)(DC)(DT)(DG)(DC)(DC)(DG)(DT) (DG) (DT)(DA)(DG)(DG)(DC)(DA)(DG)(DC) (DT)(DA)(DA)(DG)(DA)(DA)(DA)(DA)(DT)(DC) (DA)

Macromolecule #7: Prespacer, anti-sense strand of DNA

• Macromolecule: Name: Prespacer, anti-sense strand of DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: synthetic construct (others)
• Molecular weight: Theoretical: 8.263364 KDa

Sequence

String:

(DG)(DC)(DT)(DC)(DT)(DA)(DG)(DC)(DA)(DA) (DA)(DA)(DC)(DG)(DA)(DC)(DT)(DT)(DG)(DC) (DA)(DC)(DA)(DA)(DC)(DG)(DA)

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

Buffer

pH: 7.5
Component:

Concentration	Name
20.0 mM	Tris-HClTris
200.0 mM	Monopotassium glutamate
5.0 mM	EDTAEthylenediaminetetraacetic acid
1.0 mM	TCEP
0.2 %	Glycerol

• 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

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 46860 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 81000
• 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/Ų
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 5846923 / Details: Template picking
• Startup model: Type of model: OTHER; Details: The published structure of the P. aeruginosa Cas1 homodimer (PDB:3GOD) was used as starting models for the two Cas1 homodimers in this complex. The Colabfold-predicted models for the P. aeruginosa IHF heterodimer, and the P. aeruginosa Cas2/3 subunit were used as starting models. The conformation of the DNA sequences within the E. coli IHF-DNA co-crystal structure (PDB: 1IHF) was used as starting models for DNA segments within the IHFdistal and IHFproximal DNA bends. For all other double stranded DNA segments, B-form DNA was used as a starting model. Single-stranded DNA segments were built in de novo.
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
• Final 3D classification: Number classes: 4 / Avg.num./class: 275750 / Software - Name: cryoSPARC
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
• Final reconstruction: Number classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 366794
• Details: Patch motion correction and patch CTF correction were performed in cryoSPARC.