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- PDB-5czz: Crystal structure of Staphylococcus aureus Cas9 in complex with s... -

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Basic information

Entry
Database: PDB / ID: 5czz
TitleCrystal structure of Staphylococcus aureus Cas9 in complex with sgRNA and target DNA (TTGAAT PAM)
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (28-MER)
  • DNA (5'-D(*TP*TP*GP*AP*AP*TP*AP*G)-3')
  • RNA (73-MER)
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas9 / genome engineering / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
PHOSPHATE ION / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesStaphylococcus aureus subsp. aureus (bacteria)
Staphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.6 Å
AuthorsNishimasu, H. / Ishitani, R. / Nureki, O.
CitationJournal: Cell / Year: 2015
Title: Crystal Structure of Staphylococcus aureus Cas9.
Authors: Nishimasu, H. / Cong, L. / Yan, W.X. / Ran, F.A. / Zetsche, B. / Li, Y. / Kurabayashi, A. / Ishitani, R. / Zhang, F. / Nureki, O.
History
DepositionAug 1, 2015Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Sep 2, 2015Provider: repository / Type: Initial release
Revision 1.1Nov 18, 2015Group: Experimental preparation
Revision 1.2Dec 25, 2019Group: Data collection / Database references / Derived calculations
Category: citation / diffrn_source / pdbx_struct_oper_list
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _diffrn_source.pdbx_synchrotron_site / _pdbx_struct_oper_list.symmetry_operation
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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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: RNA (73-MER)
C: DNA (28-MER)
D: DNA (5'-D(*TP*TP*GP*AP*AP*TP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,16112
Polymers158,8274
Non-polymers3348
Water1,53185
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18650 Å2
ΔGint-182 kcal/mol
Surface area60380 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.585, 345.584, 98.089
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Components on special symmetry positions
IDModelComponents
11D-205-

HOH

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain DNA (28-MER)


Mass: 8469.482 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Target DNA / Source: (synth.) Staphylococcus aureus (bacteria)
#4: DNA chain DNA (5'-D(*TP*TP*GP*AP*AP*TP*AP*G)-3')


Mass: 2465.653 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Non-target DNA / Source: (synth.) Staphylococcus aureus (bacteria)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 124365.914 Da / Num. of mol.: 1 / Mutation: N580A/C946A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus subsp. aureus (bacteria)
Gene: cas9 / Plasmid: pE-SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) Rosetta2
References: UniProt: J7RUA5, Hydrolases; Acting on ester bonds
#2: RNA chain RNA (73-MER)


Mass: 23525.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Guide RNA / Source: (synth.) Staphylococcus aureus (bacteria)

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Non-polymers , 4 types, 93 molecules

#5: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Na
#6: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#7: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 85 / 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: 3.61 Å3/Da / Density % sol: 65.89 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: PEG4000, NaCl, Na2HPO4, NaN3

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 1 Å
DetectorType: PSI PILATUS 6M / Detector: PIXEL / Date: Oct 26, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.6→49.37 Å / Num. obs: 71486 / % possible obs: 99.4 % / Redundancy: 6.7 % / Net I/σ(I): 14.6
Reflection shellResolution: 2.6→2.66 Å / Redundancy: 5.6 % / Rmerge(I) obs: 0.806 / Mean I/σ(I) obs: 1.9 / % possible all: 92.8

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: SAD / Resolution: 2.6→49.044 Å / SU ML: 0.39 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 29.03 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2406 3507 4.92 %
Rwork0.2121 67814 -
obs0.2135 71321 99.19 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 260.7 Å2 / Biso mean: 92.0773 Å2 / Biso min: 34.7 Å2
Refinement stepCycle: final / Resolution: 2.6→49.044 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8419 2270 18 85 10792
Biso mean--101.14 57.96 -
Num. residues----1152
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00211164
X-RAY DIFFRACTIONf_angle_d0.47415591
X-RAY DIFFRACTIONf_chiral_restr0.021798
X-RAY DIFFRACTIONf_plane_restr0.0021605
X-RAY DIFFRACTIONf_dihedral_angle_d13.1764493
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 25

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.6-2.63560.34981340.36392427256191
2.6356-2.67330.37011350.34652536267194
2.6733-2.71320.37111360.33992718285499
2.7132-2.75560.37491330.326226372770100
2.7556-2.80070.44271510.319527142865100
2.8007-2.8490.34531330.318226902823100
2.849-2.90080.32591390.299126862825100
2.9008-2.95660.30211390.292527012840100
2.9566-3.0170.28781320.294826922824100
3.017-3.08250.33331410.288427312872100
3.0825-3.15420.33181360.2742661279799
3.1542-3.23310.28841480.267727172865100
3.2331-3.32050.32681470.2626732820100
3.3205-3.41820.24981350.244327372872100
3.4182-3.52850.24991550.228327072862100
3.5285-3.65460.24791300.215227102840100
3.6546-3.80080.26461470.213827252872100
3.8008-3.97370.22421470.205727642911100
3.9737-4.18310.26911330.192626952828100
4.1831-4.44510.20411590.181827452904100
4.4451-4.7880.17791250.169527932918100
4.788-5.26930.18721470.171927472894100
5.2693-6.03060.2181510.178727882939100
6.0306-7.59340.21371320.19852846297899
7.5934-49.05320.17921420.16162974311699
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)
11.6468-0.3108-1.73643.3626-0.43535.30760.11850.2798-0.025-1.0842-0.20320.0784-0.02140.02840.0790.66450.0891-0.08140.46740.05580.534933.392539.714169.5933
20.9835-0.9667-0.26493.4451-0.3020.58360.16050.15860.1165-0.4309-0.10240.2674-0.4715-0.0978-0.01940.97280.0759-0.20130.6050.0640.57119.912968.560773.3306
31.57290.42180.66181.85930.31351.6454-0.2981-0.06160.11170.36120.16330.2575-0.64760.18180.14530.97430.04430.18760.54610.05580.590533.130954.3706115.0382
41.0306-0.5412-0.14863.40210.03741.6196-0.0315-0.0875-0.21660.26270.06110.56620.1596-0.0768-0.04510.2994-0.035-0.03740.45440.08020.603228.587223.392493.5122
52.029-3.3267-1.38429.08982.09382.15020.29090.24430.2046-0.5804-0.2586-0.0899-0.4754-0.1002-0.03120.68410.0057-0.16250.51560.05260.50229.676647.438977.1479
61.4613-2.0703-1.09413.02041.60310.861-0.63411.695-0.9593-2.5723-0.2759-1.25831.84031.08180.50522.66130.51351.14761.6524-0.1121.822759.951511.914150.5351
71.13180.04860.4063.5608-1.29812.03340.32890.5171-0.3267-1.6237-0.20260.55120.052-0.3062-0.14511.00110.0965-0.32440.6213-0.10850.556227.831729.077864.7348
82.6332-1.65060.07267.4595-6.93358.33170.1304-0.16190.03490.3438-0.14970.37010.27430.3251-0.08170.5249-0.0511-0.03640.4148-0.0430.71337.219613.114892.0114
90.2723-0.69571.11719.3124-5.62256.7159-0.0924-0.11330.20860.5371-0.7514-0.7578-1.7484-0.15030.82570.98520.1014-0.16190.5615-0.00690.596737.327141.508375.3895
102.2861-1.3124-0.79358.65312.57272.87080.2543-0.21110.16951.379-0.0506-0.3766-0.1621-0.0965-0.20981.15140.0088-0.15940.50190.05070.63222.450964.602388.6171
110.711-0.81940.6427.9421-1.37162.58040.2165-0.2542-0.5328-0.3461-0.08780.32610.34520.0148-0.13530.4134-0.0524-0.04740.3976-0.0140.719334.150611.991889.0914
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 3 through 160 )A3 - 160
2X-RAY DIFFRACTION2chain 'A' and (resid 161 through 447 )A161 - 447
3X-RAY DIFFRACTION3chain 'A' and (resid 448 through 677 )A448 - 677
4X-RAY DIFFRACTION4chain 'A' and (resid 678 through 1052 )A678 - 1052
5X-RAY DIFFRACTION5chain 'B' and (resid 1 through 27 )B1 - 27
6X-RAY DIFFRACTION6chain 'B' and (resid 28 through 42 )B28 - 42
7X-RAY DIFFRACTION7chain 'B' and (resid 43 through 73 )B43 - 73
8X-RAY DIFFRACTION8chain 'C' and (resid 1 through 8 )C1 - 8
9X-RAY DIFFRACTION9chain 'C' and (resid 9 through 13 )C9 - 13
10X-RAY DIFFRACTION10chain 'C' and (resid 14 through 28 )C14 - 28
11X-RAY DIFFRACTION11chain 'D' and (resid 1 through 8 )D1 - 8

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