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- PDB-4rdp: Crystal structure of Cmr4 -

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Basic information

Entry
Database: PDB / ID: 4rdp
TitleCrystal structure of Cmr4
ComponentsCRISPR system Cmr subunit Cmr4
KeywordsRNA BINDING PROTEIN / RRM / Ferredoxin-like fold
Function / homologyCRISPR-associated RAMP Cmr4 / CRISPR type III-associated protein / RAMP superfamily / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / cytoplasm / CRISPR system Cmr endoribonuclease Cmr4
Function and homology information
Biological speciesPyrococcus furiosus (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.85 Å
AuthorsShao, Y. / Tang, L. / Li, H.
CitationJournal: Cell Rep / Year: 2014
Title: Essential structural and functional roles of the Cmr4 subunit in RNA cleavage by the Cmr CRISPR-Cas complex.
Authors: Nancy F Ramia / Michael Spilman / Li Tang / Yaming Shao / Joshua Elmore / Caryn Hale / Alexis Cocozaki / Nilakshee Bhattacharya / Rebecca M Terns / Michael P Terns / Hong Li / Scott M Stagg /
Abstract: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA ...The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes.
History
DepositionSep 19, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 24, 2014Provider: repository / Type: Initial release
Revision 1.1Dec 31, 2014Group: Database references
Revision 1.2Feb 28, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR system Cmr subunit Cmr4
B: CRISPR system Cmr subunit Cmr4


Theoretical massNumber of molelcules
Total (without water)67,4272
Polymers67,4272
Non-polymers00
Water0
1
A: CRISPR system Cmr subunit Cmr4


Theoretical massNumber of molelcules
Total (without water)33,7141
Polymers33,7141
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: CRISPR system Cmr subunit Cmr4


Theoretical massNumber of molelcules
Total (without water)33,7141
Polymers33,7141
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)63.035, 63.035, 189.073
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number76
Space group name H-MP41

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Components

#1: Protein CRISPR system Cmr subunit Cmr4 / CRISPR type III-B/RAMP module RAMP protein Cmr4


Mass: 33713.648 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (archaea) / Gene: cmr4 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8U1S9

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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: 2.79 Å3/Da / Density % sol: 55.84 %
Crystal growTemperature: 303 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 10% PEG8000, 6% MPD, 0.1M Tris pH8.5, VAPOR DIFFUSION, HANGING DROP, temperature 303K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 0.97937 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Jun 8, 2013
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97937 Å / Relative weight: 1
ReflectionResolution: 2.85→44.57 Å / Num. all: 17170 / Num. obs: 15822 / % possible obs: 92.15 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 2
Reflection shellResolution: 2.85→2.952 Å / % possible all: 59.55

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Processing

Software
NameVersionClassification
HKL-2000data collection
PHENIXmodel building
PHENIX(phenix.refine: 1.8.2_1309)refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.85→44.569 Å / SU ML: 0.31 / σ(F): 1.36 / Phase error: 26.12 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2349 3037 9.94 %Random
Rwork0.1797 ---
obs0.1852 30563 90.01 %-
all-30603 --
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.85→44.569 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3910 0 0 0 3910
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0133967
X-RAY DIFFRACTIONf_angle_d1.5445356
X-RAY DIFFRACTIONf_dihedral_angle_d16.0591495
X-RAY DIFFRACTIONf_chiral_restr0.065642
X-RAY DIFFRACTIONf_plane_restr0.009670
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.8502-2.89470.3464850.2729681X-RAY DIFFRACTION51
2.8947-2.94220.2979920.2604775X-RAY DIFFRACTION57
2.9422-2.99290.3422990.2465924X-RAY DIFFRACTION64
2.9929-3.04730.2785990.2282957X-RAY DIFFRACTION71
3.0473-3.10590.29211160.22521091X-RAY DIFFRACTION77
3.1059-3.16930.26391330.21691189X-RAY DIFFRACTION84
3.1693-3.23820.32451490.2191193X-RAY DIFFRACTION91
3.2382-3.31350.27011570.20851420X-RAY DIFFRACTION95
3.3135-3.39630.27431410.21071277X-RAY DIFFRACTION97
3.3963-3.48810.28911500.19931380X-RAY DIFFRACTION99
3.4881-3.59070.28221530.19361364X-RAY DIFFRACTION99
3.5907-3.70650.22351710.17381433X-RAY DIFFRACTION99
3.7065-3.83890.22861500.16321317X-RAY DIFFRACTION100
3.8389-3.99250.20111570.16171434X-RAY DIFFRACTION100
3.9925-4.17410.19111480.1581381X-RAY DIFFRACTION100
4.1741-4.3940.20141490.14741378X-RAY DIFFRACTION99
4.394-4.6690.19291470.1461361X-RAY DIFFRACTION100
4.669-5.02910.20321620.15561410X-RAY DIFFRACTION99
5.0291-5.53430.25111450.16971388X-RAY DIFFRACTION100
5.5343-6.33320.25811580.18421383X-RAY DIFFRACTION100
6.3332-7.97170.18761350.2221406X-RAY DIFFRACTION100
7.9717-44.57410.23431410.1651384X-RAY DIFFRACTION99

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