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- PDB-4un3: Crystal structure of Cas9 bound to PAM-containing DNA target -

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Basic information

Entry
Database: PDB / ID: 4un3
TitleCrystal structure of Cas9 bound to PAM-containing DNA target
Components
  • CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
  • NON-TARGET DNA STRAND
  • SGRNA
  • TARGET DNA STRAND
KeywordsHYDROLASE/DNA/RNA / HYDROLASE-DNA-RNA COMPLEX / CRISPR / 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 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain ...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 / Cas9 RuvC domain / 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 / SAD / Resolution: 2.593 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 1.3May 8, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / 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
D: NON-TARGET DNA STRAND
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,18012
Polymers197,9864
Non-polymers1948
Water7,296405
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area20040 Å2
ΔGint-225.1 kcal/mol
Surface area74660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)177.723, 68.142, 188.230
Angle α, β, γ (deg.)90.00, 111.17, 90.00
Int Tables number5
Space group name H-MC121

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain TARGET DNA STRAND


Mass: 8552.563 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#4: DNA chain NON-TARGET DNA STRAND


Mass: 3725.465 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

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RNA chain / Protein , 2 types, 2 molecules AB

#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 / CAS9


Mass: 158919.047 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

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Non-polymers , 2 types, 413 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


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


Mass: 18.015 Da / Num. of mol.: 405 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsN-TERMINAL GAAS SEQUENCE IS DERIVED FROM THE EXPRESSION VECTOR. H840A MUTATION WAS INTRODUCED TO ...N-TERMINAL GAAS SEQUENCE IS DERIVED FROM THE EXPRESSION VECTOR. H840A MUTATION WAS INTRODUCED TO INACTIVATE THE HNH 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% (W/V) PEG 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: Nov 28, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.59→48.15 Å / Num. obs: 126432 / % possible obs: 99.3 % / Observed criterion σ(I): 2 / Redundancy: 6.8 % / Rmerge(I) obs: 0.11 / Net I/σ(I): 16.3
Reflection shellResolution: 2.59→2.69 Å / Redundancy: 6.3 % / Rmerge(I) obs: 0.75 / Mean I/σ(I) obs: 2.4 / % possible all: 93.5

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE: 1.8.2_1309)refinement
XDSdata reduction
SCALAdata scaling
autoSHARPphasing
RefinementMethod to determine structure: SAD
Starting model: NONE

Resolution: 2.593→48.154 Å / SU ML: 0.39 / σ(F): 1.01 / Phase error: 28.73 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.252 6332 5 %
Rwork0.2171 --
obs0.2189 65224 98.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.593→48.154 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10690 2526 8 405 13629
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0113718
X-RAY DIFFRACTIONf_angle_d0.77919008
X-RAY DIFFRACTIONf_dihedral_angle_d19.1345552
X-RAY DIFFRACTIONf_chiral_restr0.0522171
X-RAY DIFFRACTIONf_plane_restr0.0021990
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5927-2.62220.37911600.33873040X-RAY DIFFRACTION74
2.6222-2.6530.3662160.31324030X-RAY DIFFRACTION100
2.653-2.68540.34592030.30363909X-RAY DIFFRACTION100
2.6854-2.71930.3932180.3044197X-RAY DIFFRACTION100
2.7193-2.75510.38292080.29583964X-RAY DIFFRACTION100
2.7551-2.79290.33472140.29034067X-RAY DIFFRACTION100
2.7929-2.83280.34452170.29494084X-RAY DIFFRACTION100
2.8328-2.8750.31492130.2814018X-RAY DIFFRACTION100
2.875-2.920.33352110.28273985X-RAY DIFFRACTION100
2.92-2.96780.3712200.30674093X-RAY DIFFRACTION100
2.9678-3.0190.34312100.29934001X-RAY DIFFRACTION100
3.019-3.07390.32942170.2824087X-RAY DIFFRACTION100
3.0739-3.1330.31442100.25583989X-RAY DIFFRACTION100
3.133-3.19690.28362090.25024026X-RAY DIFFRACTION100
3.1969-3.26640.25772180.2294073X-RAY DIFFRACTION100
3.2664-3.34240.2592140.22854005X-RAY DIFFRACTION100
3.3424-3.4260.29662110.23144059X-RAY DIFFRACTION100
3.426-3.51860.24182140.2294047X-RAY DIFFRACTION100
3.5186-3.62210.25682170.21773998X-RAY DIFFRACTION100
3.6221-3.73890.26632100.20764021X-RAY DIFFRACTION100
3.7389-3.87250.19162130.19184061X-RAY DIFFRACTION100
3.8725-4.02750.20542110.18224066X-RAY DIFFRACTION100
4.0275-4.21070.24082140.1864016X-RAY DIFFRACTION100
4.2107-4.43250.19182130.18154016X-RAY DIFFRACTION100
4.4325-4.710.21972110.16914073X-RAY DIFFRACTION100
4.71-5.07330.2022110.16614022X-RAY DIFFRACTION100
5.0733-5.58320.21632150.18244081X-RAY DIFFRACTION100
5.5832-6.38950.24842130.19454018X-RAY DIFFRACTION100
6.3895-8.0440.20312140.19414033X-RAY DIFFRACTION100
8.044-48.16190.19622070.18534022X-RAY DIFFRACTION99
Refinement TLS params.

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

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.09080.08160.00370.0702-0.00550.12090.01650.0316-0.01460.0247-0.0268-0.03660.00240.1257-0.0036-0.0256-0.1443-0.03950.1632-0.0250.031495.259463.611937.4695
20.01050.0011-0.00060.0094-0.00560.0051-0.00240.039-0.02730.0232-0.03110.039-0.035-0.003800.5292-0.03960.01820.3181-0.08060.5123114.676656.096266.2952
30.1885-0.10650.02320.27690.06640.3458-0.0087-0.0443-0.02450.12160.0226-0.04110.03930.1886-0.06990.1398-0.0147-0.09150.092-0.00370.1421100.082964.639355.117
40.07610.05440.04710.1312-0.02910.1193-0.00980.023-0.08050.12530.0618-0.05530.0586-0.00650.03620.18050.0397-0.02460.0622-0.03950.143282.216366.430666.9158
50.01940.00080.00320.0115-0.00240.0129-0.0209-0.0934-0.08020.1264-0.03250.02310.1004-0.051100.39640.026-0.06050.3176-0.00380.378583.983538.654872.9726
60.03240.00120.00530.03240.00960.01940.0141-0.0467-0.01480.0503-0.0344-0.05540.0298-0.02410.00390.1156-0.03290.02910.07430.00090.118255.523650.921362.3258
70.01850.0191-0.00240.06130.05680.0492-0.07460.0158-0.07770.1344-0.02290.08850.2225-0.1059-0.17240.0121-0.30330.00570.0415-0.0720.074360.573544.202137.1524
80.0643-0.04170.04630.099-0.07570.05490.02190.03110.04150.0290.0504-0.00390.08080.03970.02410.1525-0.00290.01880.1895-0.05820.130592.561360.041742.5298
90.0012-0.0011-0.00020.00190.00080.00110.00750.0167-0.0039-0.01650.00380.0485-0.0008-0.00430.00010.61720.0727-0.06830.5789-0.11610.4901109.760938.67312.1638
100.0766-0.00940.0660.04520.02360.1090.01560.04690.0135-0.02180.02230.00980.02250.06440.07060.0869-0.0523-0.01760.1324-0.02190.145785.715766.902129.9779
110.0040.0039-0.00010.0027-0.00040.0001-0.009-0.01370.03160.00530.0018-0.0377-0.02450.0367-0.00010.7267-0.0350.0320.68940.02750.733150.925272.298736.7413
120.17290.01030.10760.0739-0.01340.07290.01530.0845-0.1901-0.10910.00860.19360.1289-0.09690.01310.4597-0.0813-0.03780.4863-0.1430.368579.510842.467725.1308
130.029-0.01460.00310.03860.02590.0291-0.0095-0.07340.00580.06080.07220.04330.0066-0.01560.01190.2348-0.03790.01820.22490.03220.206381.558862.55365.6599
140.02-0.002-0.00260.0248-0.01310.00650.0106-0.028-0.20960.02380.0007-0.07860.1120.0553-0.00010.5127-0.0410.04330.479-0.10290.386977.630943.168325.8268
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1CHAIN 'B' AND (RESID 4 THROUGH 207 )
2X-RAY DIFFRACTION2CHAIN 'B' AND (RESID 208 THROUGH 299 )
3X-RAY DIFFRACTION3CHAIN 'B' AND (RESID 300 THROUGH 601 )
4X-RAY DIFFRACTION4CHAIN 'B' AND (RESID 602 THROUGH 829 )
5X-RAY DIFFRACTION5CHAIN 'B' AND (RESID 830 THROUGH 909 )
6X-RAY DIFFRACTION6CHAIN 'B' AND (RESID 910 THROUGH 1000 )
7X-RAY DIFFRACTION7CHAIN 'B' AND (RESID 1001 THROUGH 1364 )
8X-RAY DIFFRACTION8CHAIN 'A' AND (RESID 1 THROUGH 32 )
9X-RAY DIFFRACTION9CHAIN 'A' AND (RESID 33 THROUGH 40 )
10X-RAY DIFFRACTION10CHAIN 'A' AND (RESID 41 THROUGH 70 )
11X-RAY DIFFRACTION11CHAIN 'A' AND (RESID 71 THROUGH 81 )
12X-RAY DIFFRACTION12CHAIN 'C' AND (RESID 1 THROUGH 10 )
13X-RAY DIFFRACTION13CHAIN 'C' AND (RESID 11 THROUGH 28 )
14X-RAY DIFFRACTION14CHAIN 'D' AND (RESID 2 THROUGH 12 )

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