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- PDB-7cr6: Synechocystis Cas1-Cas2/prespacer binary complex -

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Basic information

Entry
Database: PDB / ID: 7cr6
TitleSynechocystis Cas1-Cas2/prespacer binary complex
Components
  • (DNA (36-MER)) x 2
  • CRISPR-associated endonuclease Cas1
  • CRISPR-associated endoribonuclease Cas2 1
KeywordsIMMUNE SYSTEM/DNA / CRISPR / adaptation / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / metal ion binding
Similarity search - Function
CRISPR-associated protein Cas1, cyanobacteria-type / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / CRISPR-associated endonuclease Cas1, N-terminal domain / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1
Similarity search - Domain/homology
DNA / DNA (> 10) / CRISPR-associated endoribonuclease Cas2 1 / CRISPR-associated endonuclease Cas1
Similarity search - Component
Biological speciesSynechocystis sp. (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.72 Å
AuthorsYu, Y. / Chen, Q.
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis.
Authors: Chengyong Wu / Dongmei Tang / Jie Cheng / Daojun Hu / Zejing Yang / Xue Ma / Haihuai He / Shaohua Yao / Tian-Min Fu / Yamei Yu / Qiang Chen /
Abstract: CRISPR-Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as ...CRISPR-Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as adaptation in the field. Some CRISPR-Cas systems rely on Cas1 and Cas2 to complete the adaptation process, which has been characterized in a few systems. In contrast, many other CRISPR-Cas systems require an additional factor of Cas4 for efficient adaptation, the mechanism of which remains less understood. Here we present biochemical reconstitution of the Synechocystis sp. PCC6803 type I-D adaptation system, X-ray crystal structures of Cas1-Cas2-prespacer complexes, and negative stained electron microscopy structure of the Cas4-Cas1 complex. Cas4 and Cas2 compete with each other to interact with Cas1. In the absence of prespacer, Cas4 but not Cas2 assembles with Cas1 into a very stable complex for processing the prespacer. Strikingly, the Cas1-prespacer complex develops a higher binding affinity toward Cas2 to form the Cas1-Cas2-prespacer ternary complex for integration. Together, we show a two-step sequential assembly mechanism for the type I-D adaptation module of Synechocystis, in which Cas4-Cas1 and Cas1-Cas2 function as two exclusive complexes for prespacer processing, capture, and integration.
History
DepositionAug 12, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 31, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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: CRISPR-associated endoribonuclease Cas2 1
F: CRISPR-associated endoribonuclease Cas2 1
G: DNA (36-MER)
H: DNA (36-MER)


Theoretical massNumber of molelcules
Total (without water)196,5838
Polymers196,5838
Non-polymers00
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area20510 Å2
ΔGint-124 kcal/mol
Surface area73510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)125.556, 215.160, 191.086
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2

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Components

#1: Protein
CRISPR-associated endonuclease Cas1


Mass: 37811.238 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. (strain PCC 6803 / Kazusa) (bacteria)
Strain: PCC 6803 / Kazusa / Gene: cas1, slr7016 / Production host: Escherichia coli (E. coli)
References: UniProt: Q6ZEI2, Hydrolases; Acting on ester bonds
#2: Protein CRISPR-associated endoribonuclease Cas2 1


Mass: 11656.425 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. (strain PCC 6803 / Kazusa) (bacteria)
Strain: PCC 6803 / Kazusa / Gene: cas2-1, ssr7017 / Production host: Escherichia coli (E. coli)
References: UniProt: Q6ZEI1, Hydrolases; Acting on ester bonds
#3: DNA chain DNA (36-MER)


Mass: 10960.986 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (36-MER)


Mass: 11064.108 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.32 Å3/Da / Density % sol: 62.99 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 30% MPD, 0.1 M imidazole pH 6.5, 0.2 M (NH4)2SO4, and 10% (w/v) PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.9793 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: May 10, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 3.7→50 Å / Num. obs: 27252 / % possible obs: 99.4 % / Redundancy: 12.5 % / Biso Wilson estimate: 44.65 Å2 / CC1/2: 0.983 / Net I/σ(I): 11
Reflection shellResolution: 3.7→3.79 Å / Num. unique obs: 1782 / CC1/2: 0.521

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
HKL-2000data scaling
PDB_EXTRACT3.25data extraction
HKL-3000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1ZPW

1zpw


Resolution: 3.72→33.81 Å / SU ML: 0.4946 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 33.0009
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3242 1056 4.19 %
Rwork0.2759 24166 -
obs0.278 25222 91.86 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 43.12 Å2
Refinement stepCycle: LAST / Resolution: 3.72→33.81 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11810 1042 0 2 12854
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002913235
X-RAY DIFFRACTIONf_angle_d0.670518113
X-RAY DIFFRACTIONf_chiral_restr0.05922009
X-RAY DIFFRACTIONf_plane_restr0.00442145
X-RAY DIFFRACTIONf_dihedral_angle_d11.18685022
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.72-3.890.3204650.30361695X-RAY DIFFRACTION51.9
3.89-4.10.41271180.2952857X-RAY DIFFRACTION88.07
4.1-4.350.30471280.2753221X-RAY DIFFRACTION98.65
4.35-4.690.30151240.26143244X-RAY DIFFRACTION99.23
4.69-5.160.35831500.26753268X-RAY DIFFRACTION99.53
5.16-5.90.31651470.2943261X-RAY DIFFRACTION99.59
5.9-7.420.37011690.30963277X-RAY DIFFRACTION99.6
7.42-33.810.26321550.24333343X-RAY DIFFRACTION97.74

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