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- PDB-6ifn: Crystal structure of Type III-A CRISPR Csm complex -

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Basic information

Entry
Database: PDB / ID: 6ifn
TitleCrystal structure of Type III-A CRISPR Csm complex
Components
  • (Type III-A CRISPR-associated RAMP protein ...) x 3
  • (Type III-A CRISPR-associated protein ...) x 2
  • RNA (32-MER)
KeywordsRNA BINDING PROTEIN / Csm complex / Type III-A / CRISPR-Cas system
Function / homology
Function and homology information


exonuclease activity / transferase activity / endonuclease activity / defense response to virus / RNA binding / ATP binding
Similarity search - Function
Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B ...Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / HD domain / HD domain
Similarity search - Domain/homology
: / RNA / RNA (> 10) / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5 / CRISPR system Cms protein Csm2 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
Similarity search - Component
Biological speciesStreptococcus thermophilus ND03 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.9 Å
AuthorsYou, L. / Wang, J. / Wang, Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China31630015 China
National Natural Science Foundation of China91440201 China
CitationJournal: Cell / Year: 2019
Title: Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference.
Authors: Lilan You / Jun Ma / Jiuyu Wang / Daria Artamonova / Min Wang / Liang Liu / Hua Xiang / Konstantin Severinov / Xinzheng Zhang / Yanli Wang /
Abstract: Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, ...Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.
History
DepositionSep 20, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 12, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 19, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.year
Revision 1.2Jan 23, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Nov 22, 2023Group: 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 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type III-A CRISPR-associated protein Csm1
F: Type III-A CRISPR-associated RAMP protein Csm3
B: Type III-A CRISPR-associated RAMP protein Csm4
E: Type III-A CRISPR-associated RAMP protein Csm3
C: Type III-A CRISPR-associated protein Csm2
D: Type III-A CRISPR-associated protein Csm2
G: Type III-A CRISPR-associated RAMP protein Csm3
H: Type III-A CRISPR-associated RAMP protein Csm5
N: RNA (32-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)278,12111
Polymers278,0019
Non-polymers1202
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area35160 Å2
ΔGint-204 kcal/mol
Surface area86780 Å2
MethodPISA
Unit cell
Length a, b, c (Å)123.543, 82.334, 161.358
Angle α, β, γ (deg.)90.00, 99.32, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11F
21E
12F
22G
13E
23G
14C
24D

NCS domain segments:

Component-ID: 0 / Refine code: 0

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11METMETALAALAFB1 - 2181 - 218
21METMETALAALAED1 - 2181 - 218
12METMETGLUGLUFB1 - 2191 - 219
22METMETVALVALGG1 - 2201 - 220
13METMETALAALAED1 - 2181 - 218
23METMETALAALAGG1 - 2181 - 218
14LEULEUGLYGLYCE4 - 1244 - 124
24THRTHRGLYGLYDF5 - 1245 - 124

NCS ensembles :
ID
1
2
3
4

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Components

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Type III-A CRISPR-associated protein ... , 2 types, 3 molecules ACD

#1: Protein Type III-A CRISPR-associated protein Csm1


Mass: 86977.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M0F3
#4: Protein Type III-A CRISPR-associated protein Csm2


Mass: 14848.145 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Gene: csm2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M049

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Type III-A CRISPR-associated RAMP protein ... , 3 types, 5 molecules FEGBH

#2: Protein Type III-A CRISPR-associated RAMP protein Csm3


Mass: 24585.840 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Gene: csm3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M035
#3: Protein Type III-A CRISPR-associated RAMP protein Csm4


Mass: 33828.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Gene: csm4 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M037
#5: Protein Type III-A CRISPR-associated RAMP protein Csm5


Mass: 41102.113 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Gene: csm5 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M038

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RNA chain , 1 types, 1 molecules N

#6: RNA chain RNA (32-MER)


Mass: 12638.505 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn

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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: 2.91 Å3/Da / Density % sol: 57.78 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop
Details: 0.1 M sodium citrate tribasic dihydrate, pH 5.0, 20% w/v polyethylene glycol 20,000.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97775 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 14, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97775 Å / Relative weight: 1
ReflectionResolution: 2.9→50 Å / Num. obs: 70612 / % possible obs: 97.2 % / Redundancy: 4.5 % / Rmerge(I) obs: 0.069 / Net I/σ(I): 8
Reflection shellResolution: 2.9→3 Å / Redundancy: 3.1 % / Rmerge(I) obs: 0.446 / Num. unique obs: 6836 / % possible all: 94.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0230refinement
HKL-3000data reduction
HKL-3000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6IFL
Resolution: 2.9→48.68 Å / Cor.coef. Fo:Fc: 0.911 / Cor.coef. Fo:Fc free: 0.879 / SU B: 17.006 / SU ML: 0.316 / Cross valid method: THROUGHOUT / ESU R Free: 0.424 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.24428 3053 5 %RANDOM
Rwork0.20118 ---
obs0.20337 57987 83.77 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 51.084 Å2
Baniso -1Baniso -2Baniso -3
1--0.03 Å20 Å2-0.81 Å2
2---0.1 Å20 Å2
3---0.37 Å2
Refinement stepCycle: 1 / Resolution: 2.9→48.68 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17376 670 2 0 18048
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.01418446
X-RAY DIFFRACTIONr_bond_other_d0.0010.01716208
X-RAY DIFFRACTIONr_angle_refined_deg1.3881.6325124
X-RAY DIFFRACTIONr_angle_other_deg1.0141.6737676
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.63152266
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.18522.445904
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.706152812
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.58815102
X-RAY DIFFRACTIONr_chiral_restr0.0680.22524
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0220715
X-RAY DIFFRACTIONr_gen_planes_other0.0010.023681
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it4.5185.3329109
X-RAY DIFFRACTIONr_mcbond_other4.5185.3319108
X-RAY DIFFRACTIONr_mcangle_it7.1537.98611360
X-RAY DIFFRACTIONr_mcangle_other7.1537.98711361
X-RAY DIFFRACTIONr_scbond_it4.1065.3399337
X-RAY DIFFRACTIONr_scbond_other4.1065.3389336
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.6057.93113764
X-RAY DIFFRACTIONr_long_range_B_refined11.32197.14474121
X-RAY DIFFRACTIONr_long_range_B_other11.32197.14474122
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11F58850.17
12E58850.17
21F57040.16
22G57040.16
31E56430.17
32G56430.17
41C31170.21
42D31170.21
LS refinement shellResolution: 2.88→2.955 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.324 103 -
Rwork0.284 1583 -
obs--31.5 %

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