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- PDB-6mua: Crystal structure of Csm1-Csm4 subcomplex in the type III-A CRISP... -

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Basic information

Entry
Database: PDB / ID: 6mua
TitleCrystal structure of Csm1-Csm4 subcomplex in the type III-A CRISPR-Csm interference complex
Components
  • Uncharacterized protein Csm1
  • Uncharacterized protein Csm4
KeywordsIMMUNE SYSTEM / Type III-A CRISPR-Cas system / Csm1-Csm4 subcomplex
Function / homology
Function and homology information


exonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / ATP binding / identical protein binding
Similarity search - Function
CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / HD domain profile. / GGDEF domain profile. / GGDEF domain / HD domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm4
Similarity search - Component
Biological speciesThermococcus onnurineus (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.91 Å
AuthorsJia, N. / Patel, D.J.
CitationJournal: Mol Cell / Year: 2019
Title: Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity.
Authors: Ning Jia / Charlie Y Mo / Chongyuan Wang / Edward T Eng / Luciano A Marraffini / Dinshaw J Patel /
Abstract: Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on ...Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus Csm binary, Csm-target RNA and Csm-target RNA ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of Csm adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into Csm complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.
History
DepositionOct 22, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 9, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 30, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.2Oct 11, 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: Uncharacterized protein Csm1
B: Uncharacterized protein Csm4


Theoretical massNumber of molelcules
Total (without water)122,0962
Polymers122,0962
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2300 Å2
ΔGint-10 kcal/mol
Surface area42230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)156.433, 156.433, 187.275
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number179
Space group name H-MP6522

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Components

#1: Protein Uncharacterized protein Csm1


Mass: 89750.602 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (archaea) / Gene: TON_0893 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWB8
#2: Protein Uncharacterized protein Csm4


Mass: 32345.061 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (archaea) / Gene: TON_0896 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC1

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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: 3.06 Å3/Da / Density % sol: 59.81 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.1 M phosphate-citrate pH 4.2, 5% PEG3000, 25% 1,2-propanediol, and 10% glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 1.2051 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 11, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.2051 Å / Relative weight: 1
ReflectionResolution: 2.9→50 Å / Num. obs: 30293 / % possible obs: 100 % / Redundancy: 16.2 % / Rpim(I) all: 0.034 / Net I/σ(I): 21.5
Reflection shellResolution: 2.9→3 Å / Num. unique obs: 2973 / Rpim(I) all: 0.376

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Processing

Software
NameVersionClassification
REFMAC5.8.0222refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6MUR

6mur


Resolution: 2.91→49.44 Å / Cor.coef. Fo:Fc: 0.935 / Cor.coef. Fo:Fc free: 0.914 / SU B: 20.502 / SU ML: 0.38 / Cross valid method: THROUGHOUT / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.27772 1499 5 %RANDOM
Rwork0.22897 ---
obs0.2314 28746 99.86 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 91.563 Å2
Baniso -1Baniso -2Baniso -3
1--1.97 Å2-0.98 Å20 Å2
2---1.97 Å20 Å2
3---6.38 Å2
Refinement stepCycle: 1 / Resolution: 2.91→49.44 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7631 0 0 0 7631
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0050.0157826
X-RAY DIFFRACTIONr_bond_other_d0.0020.0177158
X-RAY DIFFRACTIONr_angle_refined_deg1.0621.74610560
X-RAY DIFFRACTIONr_angle_other_deg0.3831.6916751
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9455941
X-RAY DIFFRACTIONr_dihedral_angle_2_deg24.54516.221307
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.319151138
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.0241576
X-RAY DIFFRACTIONr_chiral_restr0.0470.2957
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0218766
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021510
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it4.5589.1883803
X-RAY DIFFRACTIONr_mcbond_other4.5589.1883804
X-RAY DIFFRACTIONr_mcangle_it7.31413.7614731
X-RAY DIFFRACTIONr_mcangle_other7.31313.7614731
X-RAY DIFFRACTIONr_scbond_it4.4089.6854022
X-RAY DIFFRACTIONr_scbond_other4.4079.6864023
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other7.35614.3235830
X-RAY DIFFRACTIONr_long_range_B_refined11.2368738
X-RAY DIFFRACTIONr_long_range_B_other11.2358739
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.91→2.985 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.378 102 -
Rwork0.316 2055 -
obs--98.94 %

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