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- PDB-8q43: Crystal structure of cA4-bound Can2 (E341A) in complex with oligo... -

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Basic information

Entry
Database: PDB / ID: 8q43
TitleCrystal structure of cA4-bound Can2 (E341A) in complex with oligo-C DNA
Components
  • Cyclic tetraadenosine monophosphate (cA4)
  • DNA (5'-D(*CP*CP*CP*CP*C)-3')
  • DUF1887 family protein
KeywordsDNA BINDING PROTEIN / Can2 / cyclic oligoadenylates / cA4 / CARF / CRISPR ancillary nuclease / Protein-DNA complex
Function / homologytRNA endonuclease-like domain superfamily / Restriction endonuclease type II-like / nucleic acid binding / metal ion binding / : / : / DNA / RNA / DUF1887 family protein
Function and homology information
Biological speciesThermoanaerobacter brockii subsp. finnii Ako-1 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.28 Å
AuthorsJungfer, K. / Sigg, A. / Jinek, M.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-CoG-820152European Union
CitationJournal: Nucleic Acids Res. / Year: 2024
Title: Substrate selectivity and catalytic activation of the type III CRISPR ancillary nuclease Can2.
Authors: Jungfer, K. / Sigg, A. / Jinek, M.
History
DepositionAug 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 22, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 13, 2023Group: 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_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jan 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DUF1887 family protein
B: DUF1887 family protein
C: DNA (5'-D(*CP*CP*CP*CP*C)-3')
D: DNA (5'-D(*CP*CP*CP*CP*C)-3')
X: Cyclic tetraadenosine monophosphate (cA4)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)107,5028
Polymers107,3375
Non-polymers1653
Water2,810156
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8560 Å2
ΔGint-31 kcal/mol
Surface area33690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.785, 78.932, 95.046
Angle α, β, γ (deg.)90.000, 94.760, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein DUF1887 family protein


Mass: 51342.438 Da / Num. of mol.: 2 / Mutation: E341A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoanaerobacter brockii subsp. finnii Ako-1 (bacteria)
Gene: Thebr_0943 / Production host: Escherichia coli (E. coli) / References: UniProt: E8URK0
#2: DNA chain DNA (5'-D(*CP*CP*CP*CP*C)-3')


Mass: 1690.133 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Thermoanaerobacter brockii subsp. finnii Ako-1 (bacteria)
#3: RNA chain Cyclic tetraadenosine monophosphate (cA4)


Type: Polycyclic / Class: Antiviral / Mass: 1271.866 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Cyclic oligoadenylates such as c-tetraAMP were found to be novel bacterial second messengers. Antiviral in context of signalling for Type III CRISPR-Cas systems.
Source: (synth.) synthetic construct (others) / References: BIRD: PRD_002431
#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: Mn
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 156 / Source method: isolated from a natural source / Formula: H2O
Compound detailsCRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages. Cyclic ...CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages. Cyclic oligoadenylate signaling was found to be essential for the type III system against the jumbo phage.
Has ligand of interestY

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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: 1.93 Å3/Da / Density % sol: 36.3 % / Description: plate-shaped crystals
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 50 mM Tris pH 8, 17.1% (v/v) PEG 550 MME, 4.3% (w/v) PEG 2000

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Data collection

DiffractionMean temperature: 80 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Apr 7, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.28→49.1 Å / Num. obs: 33741 / % possible obs: 91.2 % / Redundancy: 6.7 % / Biso Wilson estimate: 38.91 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.08559 / Rpim(I) all: 0.03553 / Rrim(I) all: 0.09281 / Net I/σ(I): 13.05
Reflection shellResolution: 2.28→2.362 Å / Redundancy: 7 % / Rmerge(I) obs: 0.6021 / Mean I/σ(I) obs: 2.96 / Num. unique obs: 3660 / CC1/2: 0.918 / Rpim(I) all: 0.244 / Rrim(I) all: 0.6503 / % possible all: 99.84

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Processing

Software
NameVersionClassification
PHENIX1.20_4459refinement
autoPROC20230726data scaling
XDS20220110data reduction
Coot0.9.8.9model building
PHASER2.7.16phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.28→49.1 Å / SU ML: 0.3298 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 26.5409
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2427 1639 4.86 %
Rwork0.203 32079 -
obs0.2049 33718 91.21 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 54.06 Å2
Refinement stepCycle: LAST / Resolution: 2.28→49.1 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6926 139 91 156 7312
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00347322
X-RAY DIFFRACTIONf_angle_d0.64499890
X-RAY DIFFRACTIONf_chiral_restr0.04231078
X-RAY DIFFRACTIONf_plane_restr0.00321204
X-RAY DIFFRACTIONf_dihedral_angle_d12.73612737
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.28-2.350.35781340.25772908X-RAY DIFFRACTION99.84
2.35-2.420.32021380.25712929X-RAY DIFFRACTION99.87
2.42-2.510.29151520.25152896X-RAY DIFFRACTION99.87
2.51-2.610.30251500.24492926X-RAY DIFFRACTION99.87
2.61-2.730.2701770.25941516X-RAY DIFFRACTION51.92
2.73-2.870.28931620.23922909X-RAY DIFFRACTION99.97
2.87-3.050.26651380.23172922X-RAY DIFFRACTION99.9
3.05-3.290.27921490.21442936X-RAY DIFFRACTION99.97
3.29-3.620.22771550.20052230X-RAY DIFFRACTION77.84
3.62-4.140.2151850.18181945X-RAY DIFFRACTION65.63
4.14-5.220.19161590.16012940X-RAY DIFFRACTION99.97
5.22-49.10.21921400.18733022X-RAY DIFFRACTION99.75
Refinement TLS params.

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

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
11.17415915504-0.9749192100190.2095866535121.1799341835-0.01177733759691.04254502868-0.480645721499-1.005115169620.05623722158330.128716897740.3263932280680.0371443599103-0.457149149414-0.580292716389-0.06529438844520.2508685431340.1461792229710.07206205141750.707923961235-0.03025372942470.1693774889191.5076771435220.298805436443.031487083
20.444620255174-0.09980507384190.3554331806870.682353934965-0.008505033979370.9616105179160.2961953028560.238600318959-0.230323413471-0.958819438613-0.0193148131910.4579315286680.445302004617-0.2058071485650.3785382934520.7426758182030.101238875286-0.07537406848960.211633188859-0.05774425086230.3700515038821.799565104351.41862055231-4.28853645685
30.986623714007-0.334796242194-0.1033044773471.40212334051-0.7666326752211.239814965610.0671276980318-0.0916799776381-0.239539164394-0.1663673372280.03759787451970.1855579891950.1842537878330.05461305112890.04477057774190.229707157998-0.0132996864053-0.02497167383540.143860726213-0.003748508428640.3022753697931.610782330874.2130277844113.9316844647
40.195925517593-0.5089805631670.09674350849970.961132828011-0.1709340376250.323694037968-0.370208428916-0.7277875256-0.187479630414-0.009805421922810.126184322255-0.3900441801030.122869894047-0.0182604634604-0.1458183528360.3311459523690.0461721020046-0.07123319149950.5119574335990.1051073672960.29068340190829.03157470119.0220354993746.4744663196
50.780756605669-0.2411182401550.1693504902840.126599928049-0.07320048623490.0540898883071-0.352468455281-1.192753614330.7730670585870.3567056499890.474130452822-0.176963815473-0.151647046813-0.3163072671620.1159011724110.5057384875550.18051237178-0.1671702604071.03152566789-0.0773163278994-0.047667272750621.989891423515.920567477953.9084624472
60.290917843711-0.07686761682140.158520923540.0260303840243-0.1639537725090.460904461453-0.0347473021745-0.0987208775597-0.0558417492137-0.0545617657566-0.0575548905556-0.02174461850050.2044111167980.182373152293-0.01258102269570.214605003223-0.0148762136331-0.04780883013790.3184465741390.0219426201370.42260303391834.802821641615.669760947426.456902958
70.7986230502330.250935389817-0.01973328417331.741772935740.608424107660.8548076160830.2124124005140.5934100530910.363835751945-0.1281072810730.0381658101957-0.233773893285-0.225266851232-0.4082921559241.863777996120.3528744259190.02007735647210.1043141699510.5982937803750.2231510901480.40065358848729.36532437819.478455862-7.67344744802
80.9705675757810.00227455538444-0.1672081659650.4713129245410.2011701687860.8392378416510.01521782163650.02920743617950.2757370962320.0301758697495-0.00570926282758-0.056199865702-0.127107435879-0.102512920647-1.62670234641E-60.236050509642-0.009413119598370.02604385681160.2075645347710.07869395282860.34665581050225.111625555820.517719577718.7223127307
90.199508805950.0304723962429-0.03792652789590.03911985523250.06670004521570.5782928638570.07721833489560.02110041711070.4181994593980.13257582407-0.107960516412-0.458090502592-0.32617052467-0.0771553639198-0.01188045749730.3734917946710.0190401433998-0.007213947817170.2597109090310.01205952346920.52597789325120.744981130129.788907128121.4118161771
10-0.005328888276120.004352470054660.003586965292350.0203624373374-0.007323083004090.003122058883260.2458925547560.06313058704380.358096915425-0.05135855819450.124100063244-0.0774870200677-0.5039559314010.356988156733-5.90588648389E-50.8082524996420.1099990146520.03316276969111.0671995008-0.05833785612480.5888333736554.9648645021910.77731744276.69337382412
11-0.003119711979160.015071308934-0.01043811035160.0362296205149-0.0124608946343-0.007804318030270.1766281002190.199168759243-0.0914459230707-0.175072477822-0.05853758767560.155950761910.48090258223-0.297586794455-1.01294292962E-50.6903838519420.09448295498190.03208105058860.6466686729440.0235456265120.86669120419324.699719401811.70218811296.68961449133
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 6 through 175 )AA6 - 1751 - 150
22chain 'A' and (resid 176 through 253 )AA176 - 253151 - 228
33chain 'A' and (resid 254 through 436 )AA254 - 436229 - 411
44chain 'B' and (resid 1 through 69 )BB1 - 691 - 69
55chain 'B' and (resid 70 through 146 )BB70 - 14670 - 141
66chain 'B' and (resid 147 through 175 )BB147 - 175142 - 170
77chain 'B' and (resid 176 through 300 )BB176 - 300171 - 291
88chain 'B' and (resid 301 through 405 )BB301 - 405292 - 396
99chain 'B' and (resid 406 through 437 )BB406 - 437397 - 428
1010chain 'C' and (resid -1 through 3 )CC-1 - 3
1111chain 'D' and (resid -1 through 3 )DD-1 - 3

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