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- PDB-6qy3: Segment of the Cas1-Cas2-Csn2-DNA filament complex from the Type ... -

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Basic information

Entry
Database: PDB / ID: 6qy3
TitleSegment of the Cas1-Cas2-Csn2-DNA filament complex from the Type II-A CRISPR-Cas system
Components
  • (CRISPR-associated ...) x 3
  • (DNA (66-MER)) x 2
KeywordsDNA BINDING PROTEIN / CRISPR / Cas / DNA binding / Nuclease / Adaptation / Spacer acquisition / Protein complex / Protein-DNA complex / Calcium / Metal-binding / Antiviral
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endodeoxyribonuclease activity / endoribonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / metal ion binding
CRISPR associated protein Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas1 / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated endonuclease Cas1, N-terminal domain / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR-associated protein Csn2 superfamily / CRISPR-associated endonuclease Cas2 / CRISPR-associated protein Cas1, NMENI subtype / CRISPR-associated protein, Csn2-type / CRISPR-associated protein Cas1
CRISPR-associated endonuclease Cas1 / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated protein Csn2
Biological speciesStreptococcus thermophilus (bacteria)
Escherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.1 Å
AuthorsWilkinson, M. / Drabavicius, G. / Silanskas, A. / Gasiunas, G. / Siksnys, V. / Wigley, D.B.
CitationJournal: Mol. Cell / Year: 2019
Title: Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.
Authors: Martin Wilkinson / Gediminas Drabavicius / Arunas Silanskas / Giedrius Gasiunas / Virginijus Siksnys / Dale B Wigley /
Abstract: CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR ...CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas1-Cas2-Csn2, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas1-Cas2-Csn2 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMar 8, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2019Provider: repository / Type: Initial release
Revision 1.1May 22, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Jul 24, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: CRISPR-associated protein Csn2
B: CRISPR-associated protein Csn2
C: CRISPR-associated protein Csn2
D: CRISPR-associated protein Csn2
E: CRISPR-associated protein Csn2
F: CRISPR-associated protein Csn2
G: CRISPR-associated protein Csn2
H: CRISPR-associated protein Csn2
I: CRISPR-associated endonuclease Cas1
J: CRISPR-associated endonuclease Cas1
K: CRISPR-associated endoribonuclease Cas2
L: CRISPR-associated endonuclease Cas1
M: CRISPR-associated endonuclease Cas1
N: CRISPR-associated endoribonuclease Cas2
O: CRISPR-associated endonuclease Cas1
P: CRISPR-associated endonuclease Cas1
Q: CRISPR-associated endoribonuclease Cas2
R: CRISPR-associated endonuclease Cas1
S: CRISPR-associated endonuclease Cas1
T: CRISPR-associated endoribonuclease Cas2
U: CRISPR-associated endonuclease Cas1
V: CRISPR-associated endonuclease Cas1
W: CRISPR-associated endoribonuclease Cas2
X: CRISPR-associated endoribonuclease Cas2
Y: CRISPR-associated endonuclease Cas1
Z: CRISPR-associated endonuclease Cas1
a: CRISPR-associated protein Csn2
b: CRISPR-associated protein Csn2
c: CRISPR-associated protein Csn2
d: CRISPR-associated protein Csn2
e: CRISPR-associated protein Csn2
f: CRISPR-associated protein Csn2
g: CRISPR-associated protein Csn2
h: CRISPR-associated protein Csn2
i: CRISPR-associated endonuclease Cas1
j: CRISPR-associated endonuclease Cas1
k: CRISPR-associated endoribonuclease Cas2
l: CRISPR-associated endonuclease Cas1
m: CRISPR-associated endonuclease Cas1
n: CRISPR-associated endoribonuclease Cas2
o: CRISPR-associated endonuclease Cas1
p: CRISPR-associated endonuclease Cas1
q: CRISPR-associated endoribonuclease Cas2
r: CRISPR-associated endonuclease Cas1
s: CRISPR-associated endonuclease Cas1
t: CRISPR-associated endoribonuclease Cas2
u: CRISPR-associated endonuclease Cas1
v: CRISPR-associated endonuclease Cas1
w: CRISPR-associated endoribonuclease Cas2
x: CRISPR-associated endoribonuclease Cas2
y: CRISPR-associated endonuclease Cas1
z: CRISPR-associated endonuclease Cas1
1: DNA (66-MER)
2: DNA (66-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,459,73770
Polymers1,459,09654
Non-polymers64116
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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CRISPR-associated ... , 3 types, 52 molecules ABCDEFGHabcdefghIJLMOPRSUVYZij...

#1: Protein
CRISPR-associated protein Csn2


Mass: 25533.473 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csn2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G3ECR4
#2: Protein ...
CRISPR-associated endonuclease Cas1


Mass: 35363.461 Da / Num. of mol.: 24 / Mutation: C-terminal Strep tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: cas1 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: G3ECR2, Hydrolases; Acting on ester bonds
#3: Protein
CRISPR-associated endoribonuclease Cas2


Mass: 13431.560 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: cas2 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: G3ECR3, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 2 molecules 12

#4: DNA chain DNA (66-MER)


Mass: 20031.752 Da / Num. of mol.: 1
Details: Mixed long fragments of DNA pulled from the cells represented as a poly-T chain.
Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria)
#5: DNA chain DNA (66-MER)


Mass: 20626.695 Da / Num. of mol.: 1
Details: Mixed long fragments of DNA pulled from the cells represented as a poly-A chain.
Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria)

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Non-polymers , 1 types, 16 molecules

#6: Chemical
ChemComp-CA / CALCIUM ION / Calcium


Mass: 40.078 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Ca

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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-IDSourceDetails
1Cas1-Cas2-Csn2-DNA monomer complexCOMPLEX1, 2, 3, 4, 50MULTIPLE SOURCES
2Minimal unit of the Cas1-Cas2-Csn2COMPLEX1, 2, 31RECOMBINANTRepresents a small proportion of the Cas1-Cas2-Csn2-DNA particles and the smallest observed assembly of the filamentous form of the complex.
3DNACOMPLEX4, 51NATURAL
Molecular weightValue: 0.53 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Streptococcus thermophilus (bacteria)1308
33Escherichia coli BL21(DE3) (bacteria)469008
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: Buffer solution was filtered
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2150 mMSodium chlorideNaClSodium chloride1
31 mMTCEP1
SpecimenConc.: 0.05 mg/ml
Details: Sample was DNaseI treated followed by gel filtration. Peak fractions were concentrated prior to making grids.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K / Details: 15 s wait time, 1 s blot time.

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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: 75000 X / Calibrated magnification: 129032 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 92.8 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4908 / Details: Images were collected in movie mode with 39 frames
Image scansSampling size: 14 µm

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10Cootmodel refinement
11REFMACmodel refinement
12PHENIXmodel refinement
13RELION3initial Euler assignment
14RELION3final Euler assignment
15RELION3classification
16RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 349809
Details: Reproductions of an initial ab initio generated 3D reconstruction were used as templates for auto picking.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 9.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66576 / Num. of class averages: 4 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Target criteria: Correlation coefficient and visual inspection
Details: Rigid-body refinement in PHENIX of the higher resolution models, only the idealised linear DNA was subjected to jelly-body refinement in Refmac
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-IDPdb chain residue range
16QXFA1
26QXFB1
36XQFI1
46XQFJ1
56XQFK1100-110

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