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- PDB-6nbu: CRISPR Complex Subunit Csm2 from Staphylococcus epidermidis RP62a -

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Basic information

Entry
Database: PDB / ID: 6nbu
TitleCRISPR Complex Subunit Csm2 from Staphylococcus epidermidis RP62a
ComponentsCRISPR-associated protein
KeywordsSTRUCTURAL PROTEIN / CRISPR
Function / homologyCRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / defense response to virus / RNA binding / CRISPR system Cms protein Csm2
Function and homology information
Biological speciesStaphylococcus epidermidis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.75 Å
AuthorsDorsey, B.W. / Huang, L. / Mondragon, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118108 United States
CitationJournal: Nucleic Acids Res / Year: 2019
Title: Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM.
Authors: Bryan W Dorsey / Lei Huang / Alfonso Mondragón /
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas ...Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.
History
DepositionDec 10, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 13, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 27, 2019Group: Data collection / Database references / Category: citation / citation_author
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
Revision 1.2May 1, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
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 protein


Theoretical massNumber of molelcules
Total (without water)18,1811
Polymers18,1811
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area8170 Å2
2
A: CRISPR-associated protein

A: CRISPR-associated protein


Theoretical massNumber of molelcules
Total (without water)36,3612
Polymers36,3612
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation7_555y,x,-z1
Buried area2000 Å2
ΔGint-12 kcal/mol
Surface area14340 Å2
MethodPISA
Unit cell
Length a, b, c (Å)77.777, 77.777, 69.472
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212

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Components

#1: Protein CRISPR-associated protein


Mass: 18180.727 Da / Num. of mol.: 1 / Fragment: Subunit Csm2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus epidermidis (strain ATCC 35984 / RP62A) (bacteria)
Strain: ATCC 35984 / RP62A / Gene: SERP2460 / Plasmid: pMCSG7 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta (DE3) / References: UniProt: Q5HK90

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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: 2.89 Å3/Da / Density % sol: 57.43 %
Crystal growTemperature: 283 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: Tris, ethanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-G / Wavelength: 0.97856 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Jun 9, 2011
RadiationMonochromator: C(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97856 Å / Relative weight: 1
ReflectionResolution: 2.75→77.78 Å / Num. obs: 5931 / % possible obs: 99.9 % / Redundancy: 6.6 % / CC1/2: 0.999 / Net I/σ(I): 22.7
Reflection shellResolution: 2.75→2.88 Å / Redundancy: 5 % / Mean I/σ(I) obs: 1.8 / Num. unique obs: 765 / CC1/2: 0.645 / % possible all: 99.8

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Processing

Software
NameVersionClassification
REFMAC5.8.0232refinement
XDSdata reduction
Aimlessdata scaling
SHARPphasing
RefinementMethod to determine structure: SAD / Resolution: 2.75→25 Å / Cor.coef. Fo:Fc: 0.939 / Cor.coef. Fo:Fc free: 0.921 / Cross valid method: THROUGHOUT / ESU R: 0.609 / ESU R Free: 0.358 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.29418 256 4.3 %RANDOM
Rwork0.24471 ---
obs0.24689 5635 99.71 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 76.195 Å2
Baniso -1Baniso -2Baniso -3
1--0.59 Å20 Å20 Å2
2---0.59 Å20 Å2
3---1.19 Å2
Refinement stepCycle: 1 / Resolution: 2.75→25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1071 0 0 0 1071
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0131092
X-RAY DIFFRACTIONr_bond_other_d0.0340.0171018
X-RAY DIFFRACTIONr_angle_refined_deg1.6891.6481463
X-RAY DIFFRACTIONr_angle_other_deg2.2221.5822366
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.5095124
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.33523.96863
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.10115212
X-RAY DIFFRACTIONr_dihedral_angle_4_deg9.987154
X-RAY DIFFRACTIONr_chiral_restr0.1160.2135
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.021188
X-RAY DIFFRACTIONr_gen_planes_other0.0050.02240
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_it4.0298.118502
X-RAY DIFFRACTIONr_mcbond_other4.0088.112501
X-RAY DIFFRACTIONr_mcangle_it5.60712.151624
X-RAY DIFFRACTIONr_mcangle_other5.60212.158625
X-RAY DIFFRACTIONr_scbond_it5.0938.459590
X-RAY DIFFRACTIONr_scbond_other5.0898.46591
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.71812.547840
X-RAY DIFFRACTIONr_long_range_B_refined7.33393.351297
X-RAY DIFFRACTIONr_long_range_B_other7.33893.4121298
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.75→2.821 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.367 22 -
Rwork0.323 387 -
obs--99.03 %

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