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- PDB-6o75: Crystal structure of Csm1-Csm4 cassette in complex with pppApA -

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Basic information

Entry
Database: PDB / ID: 6o75
TitleCrystal structure of Csm1-Csm4 cassette in complex with pppApA
Components
  • CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
  • Csm4
  • RNA (5'-D(*(ATP))-R(P*A)-3')
Keywordsimmune system/rna / Type III-A CRISPR-Cas system / Csm1-Csm4 cassette in complex with pppApA / IMMUNE SYSTEM / immune system-dna complex / immune system-rna complex
Function / homology
Function and homology information


exonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / defense response to virus / endonuclease activity / 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 / Cas10/Cmr2, second palm domain / HD domain profile. / GGDEF domain profile. / GGDEF domain / HD domain ...: / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / Cas10/Cmr2, second palm domain / HD domain profile. / GGDEF domain profile. / GGDEF domain / HD domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
: / RNA / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm4
Similarity search - Component
Biological speciesThermococcus onnurineus (archaea)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsJia, N. / Patel, D.J.
CitationJournal: Mol Cell / Year: 2019
Title: Second Messenger cA Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path.
Authors: Ning Jia / Roger Jones / George Sukenick / Dinshaw J Patel /
Abstract: Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair ...Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cA second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppA), and products (cA), to decipher mechanistic aspects of cA formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppA intermediates and subsequent cA formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cA signaling pathway.
History
DepositionMar 7, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 31, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 11, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_conn_type
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id
Revision 1.3Oct 9, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
B: Csm4
C: RNA (5'-D(*(ATP))-R(P*A)-3')
D: RNA (5'-D(*(ATP))-R(P*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,7086
Polymers123,5984
Non-polymers1102
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5960 Å2
ΔGint-29 kcal/mol
Surface area41150 Å2
MethodPISA
Unit cell
Length a, b, c (Å)156.445, 156.445, 186.415
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 CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / ssDNase Cas10 / Cyclic oligoadenylate synthase / ToCsm1


Mass: 89706.594 Da / Num. of mol.: 1 / Mutation: D589A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (archaea) / Strain: NA1 / Gene: csm1, cas10, TON_0893 / Production host: Escherichia coli (E. coli)
References: UniProt: B6YWB8, Hydrolases; Acting on ester bonds, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: 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
#3: RNA chain RNA (5'-D(*(ATP))-R(P*A)-3')


Mass: 773.414 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
Has protein modificationY

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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: 2.7 Å3/Da / Density % sol: 54.41 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.2
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: 0.9791 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 21, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 2.6→50 Å / Num. obs: 41953 / % possible obs: 100 % / Redundancy: 34.8 % / Rpim(I) all: 0.03 / Net I/σ(I): 24.3
Reflection shellResolution: 2.6→2.69 Å / Num. unique obs: 4100

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6MUA

6mua


Resolution: 2.6→49.43 Å / Cor.coef. Fo:Fc: 0.938 / Cor.coef. Fo:Fc free: 0.924 / SU B: 13.151 / SU ML: 0.274 / Cross valid method: THROUGHOUT / ESU R: 0.568 / ESU R Free: 0.318 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.27097 2104 5 %RANDOM
Rwork0.22328 ---
obs0.22572 39795 99.76 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 80.378 Å2
Baniso -1Baniso -2Baniso -3
1--0.45 Å2-0.22 Å20 Å2
2---0.45 Å20 Å2
3---1.46 Å2
Refinement stepCycle: 1 / Resolution: 2.6→49.43 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7723 0 108 0 7831
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0040.0138015
X-RAY DIFFRACTIONr_bond_other_d0.0010.0177586
X-RAY DIFFRACTIONr_angle_refined_deg1.4021.67110842
X-RAY DIFFRACTIONr_angle_other_deg1.1221.58717504
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.0735951
X-RAY DIFFRACTIONr_dihedral_angle_2_deg29.320.405444
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.721151369
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.2351576
X-RAY DIFFRACTIONr_chiral_restr0.1070.2990
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.028816
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021844
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_it4.2048.2553840
X-RAY DIFFRACTIONr_mcbond_other4.2048.2533838
X-RAY DIFFRACTIONr_mcangle_it6.64412.3624779
X-RAY DIFFRACTIONr_mcangle_other6.64312.3634779
X-RAY DIFFRACTIONr_scbond_it4.4838.9164175
X-RAY DIFFRACTIONr_scbond_other4.5158.9154175
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other7.30913.1536064
X-RAY DIFFRACTIONr_long_range_B_refined10.79893.8258719
X-RAY DIFFRACTIONr_long_range_B_other10.79993.8178719
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.599→2.667 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.337 159 -
Rwork0.327 2809 -
obs--97.41 %

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