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- PDB-6qzt: Crystal structure of Csx1 from Sulfolobus islandicus orthorhombic form -

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Basic information

Entry
Database: PDB / ID: 6qzt
TitleCrystal structure of Csx1 from Sulfolobus islandicus orthorhombic form
ComponentsCRISPR-associated (Cas) DxTHG family
KeywordsRNA BINDING PROTEIN / CRISPR ASSOCIATED PROTEIN CARF HEPN RNAse
Function / homologyCRISPR system endoribonuclease Csx1-like / CRISPR system endoribonuclease Csx1, HEPN domain / CRISPR-associated (Cas) DxTHG family
Function and homology information
Biological speciesSulfolobus islandicus REY15A (acidophilic)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.93 Å
AuthorsMolina, R. / Montoya, G. / Sofos, N. / Stella, S.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
CitationJournal: Nat Commun / Year: 2019
Title: Structure of Csx1-cOA complex reveals the basis of RNA decay in Type III-B CRISPR-Cas.
Authors: Rafael Molina / Stefano Stella / Mingxia Feng / Nicholas Sofos / Vykintas Jauniskis / Irina Pozdnyakova / Blanca López-Méndez / Qunxin She / Guillermo Montoya /
Abstract: Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the ...Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA binding site and a ssRNA catalytic pocket. cOA undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5'-C-C-3'. The degradation of cOA by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.
History
DepositionMar 12, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated (Cas) DxTHG family
B: CRISPR-associated (Cas) DxTHG family
C: CRISPR-associated (Cas) DxTHG family


Theoretical massNumber of molelcules
Total (without water)155,5783
Polymers155,5783
Non-polymers00
Water4,756264
1
A: CRISPR-associated (Cas) DxTHG family
B: CRISPR-associated (Cas) DxTHG family

A: CRISPR-associated (Cas) DxTHG family
B: CRISPR-associated (Cas) DxTHG family

C: CRISPR-associated (Cas) DxTHG family

C: CRISPR-associated (Cas) DxTHG family


Theoretical massNumber of molelcules
Total (without water)311,1566
Polymers311,1566
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation6_445-x-1,-y-1/2,z1
crystal symmetry operation3_445-x-1,y-1/2,-z+1/21
crystal symmetry operation8_555x,-y,-z+1/21
Buried area43420 Å2
ΔGint-237 kcal/mol
Surface area106470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)107.230, 118.680, 356.680
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number24
Space group name H-MI212121

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Components

#1: Protein CRISPR-associated (Cas) DxTHG family


Mass: 51859.395 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus islandicus REY15A (acidophilic)
Gene: SiRe_0884 / Production host: Sulfolobus islandicus (acidophilic) / References: UniProt: F0NE21
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 264 / 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: 3.51 Å3/Da / Density % sol: 64.96 % / Description: Plates
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 0.1M tricine pH 8.0 0.35M NaCl 28% PEG 1000 10% Glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 3, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.79→112.74 Å / Num. obs: 38474 / % possible obs: 88.2 % / Redundancy: 14.1 % / Biso Wilson estimate: 91.13 Å2 / CC1/2: 0.99 / Net I/σ(I): 13.6
Reflection shellResolution: 2.79→3.03 Å / Redundancy: 13.1 % / Mean I/σ(I) obs: 1.7 / Num. unique obs: 1892 / CC1/2: 0.68 / % possible all: 68.6

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: SAD / Resolution: 2.93→39.32 Å / Cor.coef. Fo:Fc: 0.936 / Cor.coef. Fo:Fc free: 0.909 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.46
RfactorNum. reflection% reflectionSelection details
Rfree0.245 1715 5.05 %RANDOM
Rwork0.196 ---
obs0.198 33959 68.5 %-
Displacement parametersBiso mean: 98.08 Å2
Baniso -1Baniso -2Baniso -3
1-0.1213 Å20 Å20 Å2
2---0.5206 Å20 Å2
3---0.3993 Å2
Refine analyzeLuzzati coordinate error obs: 0.41 Å
Refinement stepCycle: LAST / Resolution: 2.93→39.32 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10926 0 0 268 11194
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0111118HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.2314994HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d4053SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes1893HARMONIC5
X-RAY DIFFRACTIONt_it11118HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_omega_torsion2.51
X-RAY DIFFRACTIONt_other_torsion23.44
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion1440SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact13507SEMIHARMONIC4
LS refinement shellResolution: 2.93→3.05 Å / Total num. of bins used: 50
RfactorNum. reflection% reflection
Rfree0.3245 -3.97 %
Rwork0.2366 653 -
all0.2402 680 -
obs--11.67 %

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