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- PDB-4un5: Crystal structure of Cas9 bound to PAM-containing DNA target cont... -

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Basic information

Entry
Database: PDB / ID: 4un5
TitleCrystal structure of Cas9 bound to PAM-containing DNA target containing mismatches at positions 1-3
Components
  • (TARGET DNA STRAND ...) x 2
  • CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
  • NON-TARGET DNA STRAND
  • SGRNA
KeywordsHYDROLASE/DNA/RNA / HYDROLASE-DNA-RNA COMPLEX / GENOME EDITING / RNP / PROTEIN-RNA COMPLEX
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
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesSTREPTOCOCCUS PYOGENES (bacteria)
SYNTHETIC CONSTRUCT (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å
AuthorsAnders, C. / Niewoehner, O. / Duerst, A. / Jinek, M.
CitationJournal: Nature / Year: 2014
Title: Structural Basis of Pam-Dependent Target DNA Recognition by the Cas9 Endonuclease
Authors: Anders, C. / Niewoehner, O. / Duerst, A. / Jinek, M.
History
DepositionMay 25, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 23, 2014Provider: repository / Type: Initial release
Revision 1.1Aug 13, 2014Group: Database references
Revision 1.2Oct 1, 2014Group: Database references
Revision 1.3Jan 10, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _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_seq_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 / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: SGRNA
B: CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
C: TARGET DNA STRAND PROXIMAL FRAGMENT
D: NON-TARGET DNA STRAND
E: TARGET DNA STRAND DISTAL FRAGMENT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,77913
Polymers197,5855
Non-polymers1948
Water13,908772
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18960 Å2
ΔGint-217.1 kcal/mol
Surface area76840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)180.182, 68.072, 190.261
Angle α, β, γ (deg.)90.00, 111.37, 90.00
Int Tables number5
Space group name H-MC121

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Components

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TARGET DNA STRAND ... , 2 types, 2 molecules CE

#3: DNA chain TARGET DNA STRAND PROXIMAL FRAGMENT


Mass: 3295.203 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#5: DNA chain TARGET DNA STRAND DISTAL FRAGMENT


Mass: 5215.419 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

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RNA chain / Protein / DNA chain , 3 types, 3 molecules ABD

#1: RNA chain SGRNA


Mass: 26788.984 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#2: Protein CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1


Mass: 158875.031 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) STREPTOCOCCUS PYOGENES (bacteria) / Strain: M1 / Plasmid: PEC-K-MBP / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): ROSETTA 2
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#4: DNA chain NON-TARGET DNA STRAND


Mass: 3410.229 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

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Non-polymers , 2 types, 780 molecules

#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 772 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsN-TERMINAL GAAS SEQUENCE IS DERIVED FROM EXPRESSION PLASMID. H840A MUTATION WAS INTRODUCED TO ...N-TERMINAL GAAS SEQUENCE IS DERIVED FROM EXPRESSION PLASMID. H840A MUTATION WAS INTRODUCED TO INACTIVATE THE HNH DOMAIN. D10A MUTATION WAS INTRODUCED TO INACTIVATE THE RUVC DOMAIN.

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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: 2.68 Å3/Da / Density % sol: 54.1 % / Description: NONE
Crystal growpH: 8.5
Details: 0.1 M TRIS-ACETATE PH 8.5, 0.3 M KSCN, 15% PEG (W/V) 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1
DetectorType: DECTRIS PILATUS / Detector: PIXEL / Date: May 10, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.4→48.35 Å / Num. obs: 164018 / % possible obs: 99.9 % / Observed criterion σ(I): 2 / Redundancy: 6.6 % / Rmerge(I) obs: 0.09 / Net I/σ(I): 15.3
Reflection shellResolution: 2.4→2.49 Å / Redundancy: 6.7 % / Rmerge(I) obs: 0.37 / Mean I/σ(I) obs: 4 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE: 1.8.2_1309)refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 4UN3
Resolution: 2.4→48.349 Å / SU ML: 0.32 / σ(F): 1.08 / Phase error: 26.2 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2465 8212 5 %
Rwork0.2149 --
obs0.2165 84384 99.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.4→48.349 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10716 2447 8 772 13943
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01113657
X-RAY DIFFRACTIONf_angle_d0.68618911
X-RAY DIFFRACTIONf_dihedral_angle_d16.3955521
X-RAY DIFFRACTIONf_chiral_restr0.0482158
X-RAY DIFFRACTIONf_plane_restr0.0021991
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4-2.42730.31482730.26945194X-RAY DIFFRACTION100
2.4273-2.45580.32342730.28325139X-RAY DIFFRACTION100
2.4558-2.48580.36562670.27265149X-RAY DIFFRACTION99
2.4858-2.51720.29592840.26615331X-RAY DIFFRACTION100
2.5172-2.55040.3162690.26365116X-RAY DIFFRACTION100
2.5504-2.58530.33272760.27045192X-RAY DIFFRACTION100
2.5853-2.62220.35532780.28795286X-RAY DIFFRACTION100
2.6222-2.66140.34152870.27885133X-RAY DIFFRACTION100
2.6614-2.70290.32632620.26355209X-RAY DIFFRACTION100
2.7029-2.74730.31272610.26185280X-RAY DIFFRACTION100
2.7473-2.79460.32752750.2695105X-RAY DIFFRACTION100
2.7946-2.84540.31272840.25835272X-RAY DIFFRACTION100
2.8454-2.90020.29362910.25365201X-RAY DIFFRACTION100
2.9002-2.95930.29912680.25985244X-RAY DIFFRACTION100
2.9593-3.02370.31592690.25645147X-RAY DIFFRACTION100
3.0237-3.0940.27622800.25265266X-RAY DIFFRACTION100
3.094-3.17140.26792740.2495119X-RAY DIFFRACTION100
3.1714-3.25710.26472760.23855239X-RAY DIFFRACTION100
3.2571-3.35290.26632740.23245138X-RAY DIFFRACTION100
3.3529-3.46110.27192660.22415219X-RAY DIFFRACTION100
3.4611-3.58480.25082860.22115276X-RAY DIFFRACTION100
3.5848-3.72830.23062720.20835132X-RAY DIFFRACTION99
3.7283-3.89790.20632610.19195156X-RAY DIFFRACTION99
3.8979-4.10330.19842750.18255143X-RAY DIFFRACTION99
4.1033-4.36020.1842660.17295179X-RAY DIFFRACTION99
4.3602-4.69660.20082740.16845182X-RAY DIFFRACTION99
4.6966-5.16880.19062750.16425170X-RAY DIFFRACTION100
5.1688-5.91550.24372730.17775213X-RAY DIFFRACTION99
5.9155-7.44860.19162730.19125200X-RAY DIFFRACTION100
7.4486-48.35880.17582700.16955176X-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)
10.0133-0.00190.02380.0318-0.00960.08490.0230.09270.00890.001-0.0010.00590.0490.08970.075-0.1803-0.13310.01830.0784-0.00560.0334109.843658.5479208.7076
20.01910.0204-0.0010.0157-0.01420.0064-0.0640.0263-0.02180.018-0.0030.0377-0.0513-0.013900.2350.02670.00320.1785-0.05970.1384137.535550.0062242.307
30.0691-0.05630.0570.1220.01090.2330.029-0.0416-0.02630.08020.0495-0.04810.04590.17350.20150.0868-0.0068-0.02840.0647-0.00650.0945124.575357.865226.5669
40.02880.00850.01970.01410.00690.0619-0.0102-0.018-0.01310.09870.0579-0.0197-0.09590.00340.01870.15530.0285-0.01130.09190.01120.1322109.905572.1481258.1484
50.0305-0.00880.00030.03040.01560.0987-0.00630.0189-0.03550.04190.0237-0.05290.09860.0876-0.01070.15850.004-0.00580.1175-0.03150.1817101.8338.8388241.51
60.13080.0499-0.02520.0580.04070.0837-0.09290.1242-0.08130.14050.05980.05870.2882-0.04670.06840.0259-0.26980.063-0.09770.0070.155780.76539.5106220.4812
70.0431-0.01330.04350.0127-0.01090.03010.060.0110.02210.02550.0138-0.00180.05850.051700.1402-0.01020.030.1732-0.04060.144114.324953.9674220.102
80.0007-0.000800.00160.00120.0010.00870.00450.006-0.00010.00770.01280.0008-0.002200.62310.0577-0.06270.5939-0.08050.46132.078431.9414179.2304
90.05290.0187-0.0360.01930.02310.10590.02880.05750.0573-0.04980.05770.0070.02390.05710.01210.1036-0.0065-0.00070.1245-0.00790.1246110.005960.9197207.1628
100.00070.0010.00080.00020.000100.026-0.00040.00240.00410.0201-0.0003-0.01610.008200.3776-0.03960.02590.34250.04470.396471.280966.3353211.2261
110.0022-0.00270.00010.00250.0004-0.0002-0.02920.0090.00390.0072-0.0101-0.02580.0087-0.025800.3292-0.0527-0.02390.3146-0.05270.342998.810132.7607200.4888
120.00490.00310.00040.0047-0.00040.00050.01090.01-0.0361-0.00470.0177-0.00270.00770.003800.2896-0.07380.01840.2482-0.10620.307698.647936.3316201.4157
130.00090.00070.00040.00070.00010.0002-0.0045-0.01710.00280.0036-0.00890.0037-0.0045-0.001400.18260.01080.01260.1889-0.00520.1522115.594958.7585230.0901
140.0009-0.0005-0.00030.000900.00070.01680.0007-0.01140.00190.01170.01260.00750.011700.2278-0.06240.02310.2673-0.01610.279999.173559.0594240.4546
150.00050.0010.00090.0023-0.00020.00060.0038-0.01120.0090.00170.00550.00880.0122-0.014600.2693-0.04310.02960.23390.05430.246995.532155.2063258.1355
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1CHAIN 'B' AND (RESID 4 THROUGH 163 )
2X-RAY DIFFRACTION2CHAIN 'B' AND (RESID 164 THROUGH 284 )
3X-RAY DIFFRACTION3CHAIN 'B' AND (RESID 285 THROUGH 551 )
4X-RAY DIFFRACTION4CHAIN 'B' AND (RESID 552 THROUGH 711 )
5X-RAY DIFFRACTION5CHAIN 'B' AND (RESID 712 THROUGH 909 )
6X-RAY DIFFRACTION6CHAIN 'B' AND (RESID 910 THROUGH 1364 )
7X-RAY DIFFRACTION7CHAIN 'A' AND (RESID 0 THROUGH 32 )
8X-RAY DIFFRACTION8CHAIN 'A' AND (RESID 33 THROUGH 40 )
9X-RAY DIFFRACTION9CHAIN 'A' AND (RESID 41 THROUGH 68 )
10X-RAY DIFFRACTION10CHAIN 'A' AND (RESID 69 THROUGH 81 )
11X-RAY DIFFRACTION11CHAIN 'C' AND (RESID 1 THROUGH 9 )
12X-RAY DIFFRACTION12CHAIN 'D' AND (RESID 3 THROUGH 12 )
13X-RAY DIFFRACTION13CHAIN 'E' AND (RESID 12 THROUGH 16 )
14X-RAY DIFFRACTION14CHAIN 'E' AND (RESID 17 THROUGH 21 )
15X-RAY DIFFRACTION15CHAIN 'E' AND (RESID 22 THROUGH 28 )

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