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- PDB-8phb: Crystal structure of apo Cami1 -

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Basic information

Entry
Database: PDB / ID: 8phb
TitleCrystal structure of apo Cami1
ComponentsCRISPR-associated protein, APE2256 family
KeywordsHYDROLASE / CRISPR / cyclic oligoadenylate / RNAse / RelE-toxin
Function / homologyCRISPR system ring nuclease SSO1393-like / CRISPR-associated protein (Cas_APE2256) / CRISPR-associated protein, APE2256 family
Function and homology information
Biological speciesAllochromatium vinosum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.7 Å
AuthorsTamulaitiene, G. / Tamulaitis, G. / Mogila, I. / Keda, K.
Funding supportLithuania, 1items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-22-09Lithuania
CitationJournal: Science / Year: 2023
Title: Ribosomal stalk-captured CARF-RelE ribonuclease inhibits translation following CRISPR signaling.
Authors: Irmantas Mogila / Giedre Tamulaitiene / Konstanty Keda / Albertas Timinskas / Audrone Ruksenaite / Giedrius Sasnauskas / Česlovas Venclovas / Virginijus Siksnys / Gintautas Tamulaitis
Abstract: Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cA) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we ...Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cA) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we characterize a class of cA-dependent effector proteins named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting of a CRISPR-associated Rossmann fold sensor domain fused to winged helix-turn-helix and a RelE-family mRNA interferase domain. Upon activation by cyclic tetra-adenylate (cA), Cami1 cleaves mRNA exposed at the ribosomal A-site thereby depleting mRNA and leading to cell growth arrest. The structures of apo-Cami1 and the ribosome-bound Cami1-cA complex delineate the conformational changes that lead to Cami1 activation and the mechanism of Cami1 binding to a bacterial ribosome, revealing unexpected parallels with eukaryotic ribosome-inactivating proteins.
History
DepositionJun 19, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated protein, APE2256 family
B: CRISPR-associated protein, APE2256 family


Theoretical massNumber of molelcules
Total (without water)91,8082
Polymers91,8082
Non-polymers00
Water6,612367
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6530 Å2
ΔGint-48 kcal/mol
Surface area31890 Å2
Unit cell
Length a, b, c (Å)65.238, 90.428, 65.616
Angle α, β, γ (deg.)90.00, 99.56, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein CRISPR-associated protein, APE2256 family


Mass: 45904.117 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Allochromatium vinosum (bacteria) / Strain: DSM180 / Gene: Alvin_3127 / Plasmid: pBAD/HisA / Production host: Escherichia coli (E. coli) / Strain (production host): DH10B / References: UniProt: D3RW14
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 367 / 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: 2.08 Å3/Da / Density % sol: 40.83 %
Crystal growTemperature: 292 K / Method: vapor diffusion, sitting drop
Details: 0.02 M carboxylic acids, 0.1 M MOPS/HEPES pH 7.5, 10% PEG 20K, 10% PEG400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9686 Å
DetectorType: DECTRIS EIGER2 R 1M / Detector: PIXEL / Date: Dec 7, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9686 Å / Relative weight: 1
ReflectionResolution: 1.7→64.71 Å / Num. obs: 81871 / % possible obs: 99.3 % / Redundancy: 7.1 % / Rmerge(I) obs: 0.042 / Rpim(I) all: 0.018 / Rrim(I) all: 0.049 / Net I/σ(I): 17.7 / Num. measured all: 578268
Reflection shellResolution: 1.7→1.79 Å / % possible obs: 99 % / Redundancy: 7 % / Rmerge(I) obs: 0.669 / Num. measured all: 83153 / Num. unique obs: 11877 / Rpim(I) all: 0.292 / Rrim(I) all: 0.784 / Net I/σ(I) obs: 2.5

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Processing

Software
NameVersionClassification
PHENIX(1.19.2_4158: ???)refinement
SCALA3.3.20data scaling
XDSMar 15, 2019data reduction
SHELXphasing
RefinementMethod to determine structure: SAD / Resolution: 1.7→64.71 Å / SU ML: 0.23 / Cross valid method: FREE R-VALUE / σ(F): 1.3 / Phase error: 24.75 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2147 16039 9.95 %
Rwork0.1858 --
obs0.1887 81810 99.04 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.7→64.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5754 0 0 367 6121
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0076136
X-RAY DIFFRACTIONf_angle_d0.838387
X-RAY DIFFRACTIONf_dihedral_angle_d12.732206
X-RAY DIFFRACTIONf_chiral_restr0.051957
X-RAY DIFFRACTIONf_plane_restr0.0071096
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7-1.720.35465440.32314896X-RAY DIFFRACTION99
1.72-1.740.33685270.31814754X-RAY DIFFRACTION99
1.74-1.760.29644870.30084832X-RAY DIFFRACTION98
1.76-1.780.31315480.29234798X-RAY DIFFRACTION99
1.78-1.810.30555060.27524863X-RAY DIFFRACTION99
1.81-1.830.34065270.25324846X-RAY DIFFRACTION99
1.83-1.860.26665560.23844828X-RAY DIFFRACTION99
1.86-1.890.26385140.22924841X-RAY DIFFRACTION99
1.89-1.910.24215730.21654807X-RAY DIFFRACTION99
1.91-1.950.23345410.20654796X-RAY DIFFRACTION99
1.95-1.980.2445290.20584926X-RAY DIFFRACTION99
1.98-2.020.25425610.20364788X-RAY DIFFRACTION99
2.02-2.050.24985470.20334830X-RAY DIFFRACTION99
2.05-2.10.2395020.20114896X-RAY DIFFRACTION99
2.1-2.140.23895560.19224799X-RAY DIFFRACTION99
2.14-2.190.23325030.17824886X-RAY DIFFRACTION99
2.19-2.250.2275370.18484858X-RAY DIFFRACTION99
2.25-2.310.20744900.17884792X-RAY DIFFRACTION99
2.31-2.380.22465560.17744847X-RAY DIFFRACTION99
2.38-2.450.22125110.18024791X-RAY DIFFRACTION98
2.45-2.540.22884890.18524925X-RAY DIFFRACTION99
2.54-2.640.26226070.1964809X-RAY DIFFRACTION99
2.64-2.760.22745550.19654768X-RAY DIFFRACTION100
2.76-2.910.21925400.19714890X-RAY DIFFRACTION100
2.91-3.090.24335430.19474854X-RAY DIFFRACTION99
3.09-3.330.21915400.19524827X-RAY DIFFRACTION99
3.33-3.660.19425260.18374888X-RAY DIFFRACTION100
3.66-4.190.18165500.15614842X-RAY DIFFRACTION99
4.19-5.280.15875790.14014808X-RAY DIFFRACTION99
5.28-64.710.18954950.17994872X-RAY DIFFRACTION99
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)
11.3504-0.3333-0.65950.76310.31481.0006-0.09580.1486-0.17640.0606-0.04430.12270.1403-0.2277-0.00040.2406-0.02730.00810.2565-0.04890.282-15.9088-6.5542-32.7205
20.3945-0.00440.31730.71390.01650.4769-0.0018-0.18340.12620.15510.084-0.1951-0.06530.21270.00010.2703-0.01820.0030.2353-0.00640.2815-1.635713.0171-14.7654
30.0120.2349-0.10130.5593-0.29490.5472-0.09290.0884-0.0675-0.0306-0.0694-0.07260.12690.2322-00.25820.02650.01560.44470.05090.275215.752-6.77593.0111
41.2907-0.3768-0.19671.29640.04621.4016-0.01310.08280.0284-0.04690.0498-0.0955-0.1064-0.0437-00.2408-0.0107-0.0040.1944-0.0190.21550.77716.1793-47.7546
50.33190.3544-0.14530.46050.4240.3504-0.0346-0.262-0.05550.2211-0.0296-0.07220.14830.147200.26670.0220.00620.25690.01390.271616.9946-11.4738-30.0449
60.83880.34530.46511.0477-0.15990.4806-0.0282-0.10060.10630.0897-0.05190.0956-0.0303-0.086700.2327-0.0091-0.00570.275-0.02090.235431.57427.0268-10.1134
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain A and resid 2:195)
2X-RAY DIFFRACTION2(chain A and resid 196:263)
3X-RAY DIFFRACTION3(chain A and resid 264:391)
4X-RAY DIFFRACTION4(chain B and resid 2:194)
5X-RAY DIFFRACTION5(chain B and resid 195:264)
6X-RAY DIFFRACTION6(chain B and resid 265:390)

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