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- EMDB-20129: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex in presenc... -

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Basic information

Entry
Database: EMDB / ID: EMD-20129
TitleBacillus halodurans Cas4-Cas1-Cas2 symmetrical complex in presence of target CRISPR DNA
Map dataBacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA
Sample
  • Organelle or cellular component: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA
    • Protein or peptide: Cas1
    • Protein or peptide: Cas2
    • Protein or peptide: Cas4
    • DNA: target CRISPR hairpin DNA
Biological speciesBacillus halodurans C-125 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 19.7 Å
AuthorsSashital DG / Lee H / Dhingra Y
CitationJournal: Elife / Year: 2019
Title: The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.
Authors: Hayun Lee / Yukti Dhingra / Dipali G Sashital /
Abstract: CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional ...CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.
History
DepositionApr 19, 2019-
Header (metadata) releaseMay 8, 2019-
Map releaseMay 8, 2019-
UpdateMay 8, 2019-
Current statusMay 8, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.21
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.21
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20129.png

Downloads & links

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Map

FileDownload / File: emd_20129.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.8 Å/pix.
x 80 pix.
= 304. Å		3.8 Å/pix.
x 80 pix.
= 304. Å		3.8 Å/pix.
x 80 pix.
= 304. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.21 / Movie #1: 0.21
Minimum - Maximum-0.077548474 - 0.39205563
Average (Standard dev.)0.005429223 (±0.03929943)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 304.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.83.83.8
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z304.000304.000304.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0780.3920.005

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Supplemental data

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Sample components

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Entire : Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with targe...

EntireName: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA
Components
  • Organelle or cellular component: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA
    • Protein or peptide: Cas1
    • Protein or peptide: Cas2
    • Protein or peptide: Cas4
    • DNA: target CRISPR hairpin DNA

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Supramolecule #1: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with targe...

SupramoleculeName: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Molecular weightExperimental: 280 KDa

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Macromolecule #1: Cas1

MacromoleculeName: Cas1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITF LTKNGRFLAR VVGESRGNVV LRKTQYRISE NDQESTKIAR NFITGKVYNS K WMLERMTR EHPLRVNVEQ FKATSQLLSV MMQEIRNCDS LESLRGWEGQ ...String:
MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITF LTKNGRFLAR VVGESRGNVV LRKTQYRISE NDQESTKIAR NFITGKVYNS K WMLERMTR EHPLRVNVEQ FKATSQLLSV MMQEIRNCDS LESLRGWEGQ AAINYNKVFD QM ILQQKEE FAFHGRSRRP PKDNVNAMLS FAYTLLANDV AAALETVGLD AYVGFMHQDR PGR ASLALD LMEELRGLYA DRFVLSLINR KEMTADGFYK KENGAVLMTD EARKTFLKAW QTKK QEKIT HPYLGEKMSW GLVPYVQALL LARFLRGDLD EYPPFLWK

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Macromolecule #2: Cas2

MacromoleculeName: Cas2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MLVLITYDVQ TSSMGGTKRL RKVAKACQNY GQRVQNSVFE CIVDSTQLTS LKLELTSLID EEKDSLRIY RLGNNYKTKV EHIGAKPSID LEDPLIF

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Macromolecule #3: Cas4

MacromoleculeName: Cas4 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MASNEEDRYL MLSGLQHFQF CKRQWALIHI EQQWEENVRT IEGQHLHKKA DQPFMKEKRG SKLTVRAMPI QSKNLQISGI CDVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA LRDKVVQMAK ...String:
MASNEEDRYL MLSGLQHFQF CKRQWALIHI EQQWEENVRT IEGQHLHKKA DQPFMKEKRG SKLTVRAMPI QSKNLQISGI CDVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA LRDKVVQMAK EMHHYYENRH TPKVKTGPFC NNCSLQSICL PKLMNKRSVK RYIEGRLSE

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Macromolecule #4: target CRISPR hairpin DNA

MacromoleculeName: target CRISPR hairpin DNA / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
SequenceString:
GATTTTCGCT GTCGCACTCT TCATGGGTGC GTGGATTGAA ATATTGACGA TAGTCAATAT TTCAATCCAC GCACCCATGA AGAGTGCGAC AGCGAAAATC

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.028 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
250.0 mMKClpotassium chloride
5.0 %C3H8O3glycerol
2.0 mMC4H10O2S2Dithiothreitol
2.0 mMMnCl2Manganese chloride
StainingType: NEGATIVE / Material: uranyl formate
Details: 3 uL of sample was applied to a glow-discharged copper 400-mesh continuous carbon grid for one minute at room temperature. The excess sample was blotted with Whatman filter paper, followed ...Details: 3 uL of sample was applied to a glow-discharged copper 400-mesh continuous carbon grid for one minute at room temperature. The excess sample was blotted with Whatman filter paper, followed by immediate application of 3 uL 2% (w/v) uranyl formate. The excess stain was blotted, followed by immediate application of 3 uL 2% uranyl formate. This step was repeated once more. The grids were allowed to dry for at least 5 minutes prior to imaging.
GridSupport film - Material: CARBON / Support film - topology: CONTINUOUS / Details: unspecified

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Electron microscopy

MicroscopeJEOL 2100
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 200 / Average exposure time: 0.5 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Particle selectionNumber selected: 115445
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4p6i


Details: low-pass filtered to 90 angstrom
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 19.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 18290
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)

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