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- PDB-6nbt: CRISPR Complex Subunit Csm3 from Staphylococcus epidermidis RP62a -

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Basic information

Entry
Database: PDB / ID: 6nbt
TitleCRISPR Complex Subunit Csm3 from Staphylococcus epidermidis RP62a
ComponentsCRISPR-associated protein
KeywordsRNA BINDING PROTEIN / CRISPR / RNA Binding / RNA Recognition Motif / Samarium (III) chloride
Function / homologyCRISPR-associated RAMP Csm3 / CRISPR type III-associated protein / RAMP superfamily / defense response to virus / endonuclease activity / RNA binding / SAMARIUM (III) ION / CRISPR system Cms endoribonuclease Csm3
Function and homology information
Biological speciesStaphylococcus epidermidis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.4 Å
AuthorsDorsey, B.W. / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated protein
B: CRISPR-associated protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,10018
Polymers54,0252
Non-polymers2,07516
Water1,22568
1
A: CRISPR-associated protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,9959
Polymers27,0121
Non-polymers9828
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: CRISPR-associated protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,1059
Polymers27,0121
Non-polymers1,0938
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
B: CRISPR-associated protein
hetero molecules

B: CRISPR-associated protein
hetero molecules

A: CRISPR-associated protein
hetero molecules

A: CRISPR-associated protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)112,19936
Polymers108,0504
Non-polymers4,15032
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_455-x-1,y,-z1
crystal symmetry operation3_455x-1/2,y+1/2,z1
crystal symmetry operation4_455-x-1/2,y+1/2,-z1
Buried area11510 Å2
ΔGint-188 kcal/mol
Surface area36800 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.612, 50.979, 83.018
Angle α, β, γ (deg.)90.00, 97.05, 90.00
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-431-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A2 - 214
2010B2 - 214

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Components

#1: Protein CRISPR-associated protein


Mass: 27012.383 Da / Num. of mol.: 2 / Fragment: Subunit Csm3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus epidermidis (strain ATCC 35984 / RP62A) (bacteria)
Strain: ATCC 35984 / RP62A / Cell line: 35984D-5 / Gene: SERP2459 / Plasmid: pMCSG7 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta (DE3) / References: UniProt: Q5HK91
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#3: Chemical
ChemComp-SM / SAMARIUM (III) ION / Samarium


Mass: 150.360 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: Sm
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 68 / 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: 2.03 Å3/Da / Density % sol: 39.51 %
Crystal growTemperature: 287 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: PEG 8000, calcium acetate, 2-(N-morpholino)ethanesulfonic acid (MES)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 1.0781 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Feb 28, 2018
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0781 Å / Relative weight: 1
ReflectionResolution: 2.4→46.57 Å / Num. obs: 16891 / % possible obs: 98.2 % / Redundancy: 6.8 % / Net I/σ(I): 10.3
Reflection shellResolution: 2.4→2.49 Å

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Processing

Software
NameVersionClassification
XDS20180126data reduction
Aimless0.7.3data scaling
CRANK22.0.180phasing
REFMAC5.8.0222refinement
RefinementMethod to determine structure: SAD / Resolution: 2.4→25 Å / Cor.coef. Fo:Fc: 0.939 / Cor.coef. Fo:Fc free: 0.921 / SU B: 10.897 / SU ML: 0.246 / Cross valid method: THROUGHOUT / ESU R: 0.491 / ESU R Free: 0.284 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.26687 795 4.7 %RANDOM
Rwork0.23046 ---
obs0.23226 16078 97.89 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 50.19 Å2
Baniso -1Baniso -2Baniso -3
1--0.84 Å2-0 Å21.31 Å2
2--0.02 Å20 Å2
3---0.48 Å2
Refinement stepCycle: 1 / Resolution: 2.4→25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2932 0 14 68 3014
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0142977
X-RAY DIFFRACTIONr_bond_other_d0.0010.0172694
X-RAY DIFFRACTIONr_angle_refined_deg1.2921.6643986
X-RAY DIFFRACTIONr_angle_other_deg0.8321.6566321
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.1445359
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.9722.768177
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.60515550
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.31522
X-RAY DIFFRACTIONr_chiral_restr0.0580.2391
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.023327
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02541
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_it3.9844.8521460
X-RAY DIFFRACTIONr_mcbond_other3.9794.8481459
X-RAY DIFFRACTIONr_mcangle_it6.2447.241811
X-RAY DIFFRACTIONr_mcangle_other6.2447.2441812
X-RAY DIFFRACTIONr_scbond_it4.565.4231517
X-RAY DIFFRACTIONr_scbond_other4.5595.4221518
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other7.2867.9022175
X-RAY DIFFRACTIONr_long_range_B_refined31.29456.053124
X-RAY DIFFRACTIONr_long_range_B_other29.56456.0343123
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 5423 / Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.09 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 2.4→2.462 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.399 64 -
Rwork0.305 1121 -
obs--95.64 %

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