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- PDB-4zt0: Crystal structure of catalytically-active Streptococcus pyogenes ... -

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Basic information

Entry
Database: PDB / ID: 4zt0
TitleCrystal structure of catalytically-active Streptococcus pyogenes Cas9 in complex with single-guide RNA at 2.9 Angstrom resolution
Components
  • CRISPR-associated endonuclease Cas9
  • single-guide RNA
KeywordsHYDROLASE/RNA / CRISPR-Cas9 / bacteria adaptive immunity / DNA endonuclease / HYDROLASE-RNA complex / genome editing and regulation
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / : / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.9 Å
AuthorsJiang, F. / Doudna, J.A.
CitationJournal: Science / Year: 2015
Title: A Cas9-guide RNA complex preorganized for target DNA recognition.
Authors: Jiang, F. / Zhou, K. / Ma, L. / Gressel, S. / Doudna, J.A.
History
DepositionMay 14, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 8, 2015Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: single-guide RNA
C: CRISPR-associated endonuclease Cas9
D: single-guide RNA


Theoretical massNumber of molelcules
Total (without water)374,6124
Polymers374,6124
Non-polymers00
Water0
1
A: CRISPR-associated endonuclease Cas9
B: single-guide RNA


Theoretical massNumber of molelcules
Total (without water)187,3062
Polymers187,3062
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10840 Å2
ΔGint-103 kcal/mol
Surface area72470 Å2
MethodPISA
2
C: CRISPR-associated endonuclease Cas9
D: single-guide RNA


Theoretical massNumber of molelcules
Total (without water)187,3062
Polymers187,3062
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11100 Å2
ΔGint-99 kcal/mol
Surface area71830 Å2
MethodPISA
Unit cell
Length a, b, c (Å)103.238, 143.002, 294.917
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein CRISPR-associated endonuclease Cas9 / Csn1 protein / Cas9 / CRISPR-Cas9


Mass: 159804.594 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: csn1, cas9, ERS445054_00848, MTB314_0777 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A0C6FZC2, UniProt: Q99ZW2*PLUS, Hydrolases; Acting on ester bonds
#2: RNA chain single-guide RNA


Mass: 27501.404 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.92 Å3/Da / Density % sol: 57.9 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6
Details: 12% w/v PEG8000, 100 mM sodium cacodylate, pH 6.0, 250 mM sodium chloride

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 0.98 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Dec 5, 2014
RadiationMonochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2.9→102.7 Å / Num. all: 97517 / Num. obs: 97000 / % possible obs: 99.4 % / Redundancy: 9.8 % / Biso Wilson estimate: 75.04 Å2 / Rmerge(I) obs: 0.035 / Net I/σ(I): 12.9
Reflection shellResolution: 2.9→3 Å / Redundancy: 6.6 % / Rmerge(I) obs: 0.456 / Mean I/σ(I) obs: 1.7 / % possible all: 96.7

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: SAD / Resolution: 2.9→49.153 Å / SU ML: 0.4 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 28.81 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflectionSelection details
Rfree0.2637 4800 4.96 %SAD-MR was used
Rwork0.2382 ---
obs0.2395 96760 99.26 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.9→49.153 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20700 3090 0 0 23790
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00224530
X-RAY DIFFRACTIONf_angle_d0.55433892
X-RAY DIFFRACTIONf_dihedral_angle_d12.0699424
X-RAY DIFFRACTIONf_chiral_restr0.0233968
X-RAY DIFFRACTIONf_plane_restr0.0023831
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.9-2.9330.37061640.35142826X-RAY DIFFRACTION93
2.933-2.96750.37061360.34262912X-RAY DIFFRACTION95
2.9675-3.00370.34751380.32552965X-RAY DIFFRACTION97
3.0037-3.04170.3841630.31933015X-RAY DIFFRACTION97
3.0417-3.08170.35591500.32322950X-RAY DIFFRACTION99
3.0817-3.12390.36561570.32233076X-RAY DIFFRACTION99
3.1239-3.16850.35781540.31832998X-RAY DIFFRACTION99
3.1685-3.21580.34281770.31333064X-RAY DIFFRACTION100
3.2158-3.2660.30471450.28793044X-RAY DIFFRACTION100
3.266-3.31960.28731360.27453098X-RAY DIFFRACTION100
3.3196-3.37680.31431680.27663020X-RAY DIFFRACTION100
3.3768-3.43820.27311590.27643085X-RAY DIFFRACTION100
3.4382-3.50430.3251470.25923043X-RAY DIFFRACTION100
3.5043-3.57580.31161650.25993092X-RAY DIFFRACTION100
3.5758-3.65350.28451610.25073042X-RAY DIFFRACTION100
3.6535-3.73850.2751620.25143071X-RAY DIFFRACTION100
3.7385-3.8320.26991660.23833089X-RAY DIFFRACTION100
3.832-3.93550.24291720.22663028X-RAY DIFFRACTION100
3.9355-4.05130.23041850.22423071X-RAY DIFFRACTION100
4.0513-4.1820.24731630.21823064X-RAY DIFFRACTION100
4.182-4.33140.24691520.21163115X-RAY DIFFRACTION100
4.3314-4.50470.20351530.20673099X-RAY DIFFRACTION100
4.5047-4.70950.19371500.19453082X-RAY DIFFRACTION100
4.7095-4.95760.22121460.20273130X-RAY DIFFRACTION100
4.9576-5.26790.25091660.2023088X-RAY DIFFRACTION100
5.2679-5.67410.27781870.22473115X-RAY DIFFRACTION100
5.6741-6.24410.27111640.2333128X-RAY DIFFRACTION100
6.2441-7.14520.25591580.23913166X-RAY DIFFRACTION100
7.1452-8.99320.21731670.21213192X-RAY DIFFRACTION100
8.9932-49.15990.25111890.22683292X-RAY DIFFRACTION99
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)
10.42680.3120.12921.4205-0.42960.5619-0.2213-0.0072-0.1818-0.1668-0.3849-0.71330.39910.3369-0.41150.49180.28730.15660.62430.65960.851241.76-4.2478-40.5455
21.3225-0.4030.6331.7162-0.35941.7197-0.20590.33070.3377-0.7795-0.3913-0.58810.34120.02280.08150.68190.12150.17020.61710.1810.676435.4728-0.1983-57.1491
30.2159-0.6645-0.15154.7488-1.51791.84540.11460.1786-0.0468-0.7139-0.05960.2055-0.01290.0901-0.04020.5629-0.0804-0.09090.6380.13010.55870.058715.537-46.1095
41.763-0.48060.15122.3799-0.51361.5282-0.2033-0.17940.16550.4590.10370.1419-0.1660.24250.12060.482-0.05420.03360.43540.05120.37048.29186.5164-18.8585
56.97160.82040.47811.66711.15692.1217-0.4529-0.39550.4024-0.3363-0.177-0.9981-0.31480.40820.19220.7046-0.03570.03840.85320.4821.011747.1143-6.3807-33.7753
63.1247-0.02670.59891.31170.51550.32010.3936-1.7935-0.56690.1653-0.42440.4740.3735-0.3881-0.2711.7825-0.7274-1.15073.22980.66792.953474.03235.0047-4.0826
70.8173-0.2589-1.90135.25544.46137.2820.2725-0.0416-0.27360.851.1362-1.0680.32491.4519-1.11080.9822-0.0764-0.58921.36880.48792.136469.19774.4133-24.3621
80.3909-0.54310.25221.19550.07840.7916-0.2543-0.3353-0.52460.0314-0.2736-0.42390.36980.213-0.02250.68520.0990.22350.64430.39050.776534.2704-17.5727-30.7422
98.6079-1.67131.16498.41410.8418.41250.37050.705-0.4279-0.0463-0.25570.08360.59270.1844-0.12290.7430.07080.16370.40990.0810.62853.5676-23.1544-22.168
105.3712-0.65571.81410.8331-0.26831.5836-0.04260.2630.02540.2044-0.07270.0814-0.1391-0.08430.11930.7766-0.0052-0.03650.4466-0.05630.327415.3563-53.9147-29.0622
110.9806-1.3957-0.65345.46791.43781.6471-0.04150.0946-0.2086-0.5407-0.0402-0.1381-0.0333-0.13980.06810.5172-0.10320.03050.3849-0.13970.40146.1833-75.0843-52.8551
123.6060.8299-3.3012.7834-1.25825.2039-0.5640.3081-1.13420.2474-0.07150.59380.0682-0.43620.54630.6523-0.05340.13880.5993-0.06250.8421-7.1071-74.4509-37.2102
132.2076-0.1692-0.22571.7521-0.53361.612-0.24760.6654-0.1459-0.15080.09620.37620.1476-0.29310.06550.5606-0.02930.02020.5302-0.16610.4392-5.5213-59.6677-44.5666
144.078-2.1691-3.0224.94934.08835.8544-0.32480.7494-0.4718-0.4126-0.05010.08410.0263-0.08680.21920.693-0.21770.14930.9126-0.06170.552226.4197-62.8746-66.4253
151.47621.03961.94791.02410.39374.932-0.18380.6554-0.404-0.23130.2427-0.3926-0.55290.7088-0.07220.65070.06110.00981.153-0.09610.705244.5602-61.2544-53.4886
161.2719-1.51490.11522.5628-0.17172.63870.34070.9671-1.2276-0.5664-0.2710.19940.97570.2142-0.06141.25570.1827-0.17321.7707-0.52111.455849.8154-88.6219-43.7345
172.6052-0.111.11380.6845-0.851.2247-0.18890.1876-0.47830.16180.0359-0.20780.00020.1086-0.0850.78190.098-0.160.6456-0.17990.584544.2556-68.8268-19.5998
184.2788-1.49070.46662.06241.98964.191-0.1709-0.5358-0.58950.7281-0.04690.8230.3198-0.85360.23420.6113-0.0887-0.11020.63450.04090.476941.0367-76.404-8.8482
195.0919-0.0352-3.13421.68740.31215.636-0.6167-0.3738-0.40940.24520.22820.19230.6197-0.10070.40170.79840.05490.01790.5769-00.43373.4951-60.1331-31.4762
203.4455-2.6756-4.40274.93472.64355.9504-0.1137-1.6802-0.13352.6990.91950.6218-0.4271-0.4146-0.74541.93450.06190.41.51970.16231.1709-19.6483-70.6472-0.6081
214.4507-0.0414-2.28951.93893.60717.8028-0.1749-0.7456-1.10420.97610.16980.64171.2527-0.29360.12131.20630.12560.57391.11380.22681.0561-17.9946-72.5378-19.2566
222.3848-0.2121.0040.8343-0.30290.4928-0.2646-0.24880.33590.33490.0896-0.0984-0.0710.0550.15860.78920.0943-0.11210.6282-0.1270.4216.6467-48.8408-29.6761
237.3942.19131.62111.3618-1.53025.98760.09351.27060.70810.4251-0.60920.0349-1.07590.4110.42681.48820.1054-0.41870.75510.13571.132248.4636-42.2518-22.2984
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 4 through 255 )
2X-RAY DIFFRACTION2chain 'A' and (resid 256 through 712 )
3X-RAY DIFFRACTION3chain 'A' and (resid 713 through 1018 )
4X-RAY DIFFRACTION4chain 'A' and (resid 1019 through 1366 )
5X-RAY DIFFRACTION5chain 'B' and (resid 11 through 30 )
6X-RAY DIFFRACTION6chain 'B' and (resid 31 through 38 )
7X-RAY DIFFRACTION7chain 'B' and (resid 39 through 45 )
8X-RAY DIFFRACTION8chain 'B' and (resid 46 through 68 )
9X-RAY DIFFRACTION9chain 'B' and (resid 69 through 82 )
10X-RAY DIFFRACTION10chain 'C' and (resid 4 through 150 )
11X-RAY DIFFRACTION11chain 'C' and (resid 151 through 283 )
12X-RAY DIFFRACTION12chain 'C' and (resid 284 through 376 )
13X-RAY DIFFRACTION13chain 'C' and (resid 377 through 455 )
14X-RAY DIFFRACTION14chain 'C' and (resid 456 through 653 )
15X-RAY DIFFRACTION15chain 'C' and (resid 654 through 750 )
16X-RAY DIFFRACTION16chain 'C' and (resid 751 through 980 )
17X-RAY DIFFRACTION17chain 'C' and (resid 981 through 1280 )
18X-RAY DIFFRACTION18chain 'C' and (resid 1281 through 1366 )
19X-RAY DIFFRACTION19chain 'D' and (resid 11 through 31 )
20X-RAY DIFFRACTION20chain 'D' and (resid 32 through 37 )
21X-RAY DIFFRACTION21chain 'D' and (resid 38 through 44 )
22X-RAY DIFFRACTION22chain 'D' and (resid 45 through 68 )
23X-RAY DIFFRACTION23chain 'D' and (resid 69 through 82 )

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