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- PDB-6qzq: Crystal structure of Csx1 from Sulfolobus islandicus monoclinic form -

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Basic information

Entry
Database: PDB / ID: 6qzq
TitleCrystal structure of Csx1 from Sulfolobus islandicus monoclinic form
ComponentsCRISPR-associated (Cas) DxTHG family
KeywordsRNA BINDING PROTEIN / CRISPR ASSOCIATED PROTEIN CARF HEPN RNAse
Function / homologyCRISPR system endoribonuclease Csx1-like / CRISPR-associated (Cas) DxTHG family / CRISPR-associated (Cas) DxTHG family
Function and homology information
Biological speciesSulfolobus islandicus (acidophilic)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3.6 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 1.0Jan 22, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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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: CRISPR-associated (Cas) DxTHG family
E: CRISPR-associated (Cas) DxTHG family
F: CRISPR-associated (Cas) DxTHG family


Theoretical massNumber of molelcules
Total (without water)311,1566
Polymers311,1566
Non-polymers00
Water0
1
A: CRISPR-associated (Cas) DxTHG family
B: CRISPR-associated (Cas) DxTHG family

E: CRISPR-associated (Cas) DxTHG family
F: CRISPR-associated (Cas) DxTHG family

C: CRISPR-associated (Cas) DxTHG family
D: CRISPR-associated (Cas) DxTHG family


Theoretical massNumber of molelcules
Total (without water)311,1566
Polymers311,1566
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y+1/2,-z1
crystal symmetry operation2_445-x-1,y-1/2,-z1
Buried area44310 Å2
ΔGint-235 kcal/mol
Surface area105930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)108.304, 119.579, 190.133
Angle α, β, γ (deg.)90.00, 106.25, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 51859.395 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus islandicus (strain REY15A) (acidophilic)
Gene: SiRe_0884 / Production host: Sulfolobus islandicus (acidophilic) / References: UniProt: F0NE21

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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: 3.65 Å3/Da / Density % sol: 66 % / Description: Plates
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5 / Details: 0.1M Tris pH=8.5 8% PEG 8000

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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: May 18, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.6→72.55 Å / Num. obs: 49216 / % possible obs: 99.5 % / Observed criterion σ(I): 2 / Redundancy: 7.9 % / CC1/2: 0.99 / Net I/σ(I): 11.2
Reflection shellResolution: 3.6→3.79 Å / Redundancy: 6.8 % / Mean I/σ(I) obs: 2.9 / Num. unique obs: 1279 / CC1/2: 0.87 / % possible all: 90.7

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Processing

Software
NameVersionClassification
PHENIX(1.14_3260: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: SAD / Resolution: 3.6→72.55 Å / SU ML: 0.48 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 26.12
RfactorNum. reflection% reflection
Rfree0.2571 2456 4.99 %
Rwork0.2004 --
obs0.2033 49216 90.73 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.6→72.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21846 0 0 0 21846
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00522230
X-RAY DIFFRACTIONf_angle_d0.95929976
X-RAY DIFFRACTIONf_dihedral_angle_d7.96213608
X-RAY DIFFRACTIONf_chiral_restr0.0513372
X-RAY DIFFRACTIONf_plane_restr0.0043852
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.6-3.66930.3924550.29531224X-RAY DIFFRACTION86
3.6693-3.74420.29631090.28182355X-RAY DIFFRACTION92
3.7442-3.82560.31681640.26182802X-RAY DIFFRACTION100
3.8256-3.91450.2478240.2793491X-RAY DIFFRACTION17
3.9145-4.01240.36271310.24252858X-RAY DIFFRACTION98
4.0124-4.12090.27911250.22572855X-RAY DIFFRACTION100
4.1209-4.24220.25381730.2142833X-RAY DIFFRACTION100
4.2422-4.37910.26021520.19742813X-RAY DIFFRACTION100
4.3791-4.53560.23451380.18572852X-RAY DIFFRACTION100
4.5356-4.71710.23911810.18182795X-RAY DIFFRACTION99
4.7171-4.93180.26781620.18442860X-RAY DIFFRACTION100
4.9318-5.19170.27521460.19192833X-RAY DIFFRACTION99
5.1917-5.51690.26531480.2052786X-RAY DIFFRACTION98
5.5169-5.94270.27341390.2192874X-RAY DIFFRACTION100
5.9427-6.54030.28711600.22322841X-RAY DIFFRACTION100
6.5403-7.48590.26731510.21012888X-RAY DIFFRACTION100
7.4859-9.42820.21271570.15542876X-RAY DIFFRACTION100
9.4282-72.56440.19781410.16472924X-RAY DIFFRACTION98
Refinement TLS params.Method: refined / Origin x: -33.8387 Å / Origin y: 26.8036 Å / Origin z: 50.4023 Å
111213212223313233
T0.6713 Å2-0.0015 Å2-0.0378 Å2-0.5447 Å2-0.0776 Å2--0.5367 Å2
L0.2695 °2-0.197 °2-0.039 °2-0.1236 °2-0.0248 °2---0.076 °2
S-0.1388 Å °-0.0773 Å °0.0112 Å °0.0502 Å °0.0886 Å °0.0624 Å °0.0205 Å °-0.0641 Å °0.0365 Å °
Refinement TLS groupSelection details: all

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