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- PDB-6r7b: Crystal structure of Csx1 in complex with cyclic oligoadenylate c... -

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Basic information

Entry
Database: PDB / ID: 6r7b
TitleCrystal structure of Csx1 in complex with cyclic oligoadenylate cOA4 conformation 1
Components
  • CRISPR-associated (Cas) DxTHG family
  • RNA (5'-R(P*AP*AP*AP*A)-3')
KeywordsRNA BINDING PROTEIN / CRISPR ASSOCIATED PROTEIN CARF HEPN RNAse
Function / homologyCRISPR system endoribonuclease Csx1-like / CRISPR system endoribonuclease Csx1, HEPN domain / RNA / CRISPR-associated (Cas) DxTHG family
Function and homology information
Biological speciesSulfolobus islandicus (acidophilic)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.12 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 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: 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: CRISPR-associated (Cas) DxTHG family
B: CRISPR-associated (Cas) DxTHG family
C: CRISPR-associated (Cas) DxTHG family
D: RNA (5'-R(P*AP*AP*AP*A)-3')
E: RNA (5'-R(P*AP*AP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)158,1225
Polymers158,1225
Non-polymers00
Water1,71195
1
A: CRISPR-associated (Cas) DxTHG family
B: CRISPR-associated (Cas) DxTHG family
D: RNA (5'-R(P*AP*AP*AP*A)-3')

A: CRISPR-associated (Cas) DxTHG family
B: CRISPR-associated (Cas) DxTHG family
D: RNA (5'-R(P*AP*AP*AP*A)-3')

C: CRISPR-associated (Cas) DxTHG family
E: RNA (5'-R(P*AP*AP*AP*A)-3')

C: CRISPR-associated (Cas) DxTHG family
E: RNA (5'-R(P*AP*AP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)316,24410
Polymers316,24410
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation6_565-x,-y+3/2,z1
crystal symmetry operation3_555-x,y+1/2,-z+1/21
crystal symmetry operation8_565x,-y+1,-z+1/21
Buried area51120 Å2
ΔGint-236 kcal/mol
Surface area103940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)107.080, 117.680, 357.260
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 (acidophilic) / Gene: SiRe_0884 / Production host: Sulfolobus islandicus (acidophilic) / References: UniProt: F0NE21
#2: RNA chain RNA (5'-R(P*AP*AP*AP*A)-3')


Mass: 1271.866 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Although Chain D and E have the same composition, due to crystal symmetry chain E is just a half of D. Then chain D contains 4 adenosine monophosphate while chain E contains 2 adenosine monophosphates
Source: (synth.) synthetic construct (others)
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 95 / 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: 3.5 Å3/Da / Density % sol: 64.85 % / Description: PLATES
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 0.1M TRICINE PH=8.0, 0.35M NACL, 28% PEG1000, 10% GLYCEROL

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 0.9785 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 3, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9785 Å / Relative weight: 1
ReflectionResolution: 3.12→178.63 Å / Num. obs: 24510 / % possible obs: 93 % / Redundancy: 11.6 % / Biso Wilson estimate: 115.32 Å2 / CC1/2: 0.99 / Net I/σ(I): 11.7
Reflection shellResolution: 3.12→3.46 Å / Redundancy: 13.1 % / Mean I/σ(I) obs: 1.4 / Num. unique obs: 1226 / CC1/2: 0.57

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6QZT

6qzt


Resolution: 3.12→48.29 Å / Cor.coef. Fo:Fc: 0.948 / Cor.coef. Fo:Fc free: 0.923 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.556
RfactorNum. reflection% reflectionSelection details
Rfree0.229 1206 4.92 %RANDOM
Rwork0.18 ---
obs0.183 24506 60.3 %-
Displacement parametersBiso mean: 131.32 Å2
Baniso -1Baniso -2Baniso -3
1--7.0887 Å20 Å20 Å2
2--2.883 Å20 Å2
3---4.2058 Å2
Refine analyzeLuzzati coordinate error obs: 0.41 Å
Refinement stepCycle: LAST / Resolution: 3.12→48.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10926 132 0 96 11154
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0111266HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.2615222HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d4079SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes1899HARMONIC5
X-RAY DIFFRACTIONt_it11266HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_omega_torsion2.52
X-RAY DIFFRACTIONt_other_torsion24.99
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion1464SEMIHARMONIC5
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_contact13450SEMIHARMONIC4
LS refinement shellResolution: 3.12→3.33 Å / Total num. of bins used: 50
RfactorNum. reflection% reflection
Rfree0.3216 -4.28 %
Rwork0.2217 470 -
all0.226 491 -
obs--6.92 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.35320.72190.89551.9350.35991.405-0.09780.08630.2386-0.080.06770.41730.0986-0.22290.0301-0.19810.152-0.152-0.17190.0163-0.171-42.65764.887823.9839
22.66990.86110.66761.19690.39690.737-0.01010.2213-0.03220.14120.02210.19340.1611-0.1462-0.0120.04350.152-0.152-0.1161-0.0061-0.0955-39.938559.538624.7608
30.97240.0841-0.20631.33040.46723.49930.1014-0.13740.03430.0647-0.166-0.1938-0.07470.22560.0646-0.3040.0525-0.0749-0.1710.0055-0.08252.086131.420368.5466
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{ A|* }
2X-RAY DIFFRACTION2{ B|* }
3X-RAY DIFFRACTION3{ C|* }

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