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- PDB-4cmp: Crystal structure of S. pyogenes Cas9 -

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Basic information

Entry
Database: PDB / ID: 4cmp
TitleCrystal structure of S. pyogenes Cas9
ComponentsCRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
KeywordsHYDROLASE / DNASE / RNA-GUIDED / IMMUNITY / CRRNA / GENOME EDITING
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 / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...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 / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesSTREPTOCOCCUS PYOGENES (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MIRAS / Resolution: 2.62 Å
AuthorsJinek, M. / Jiang, F. / Taylor, D.W. / Sternberg, S.H. / Kaya, E. / Ma, E. / Anders, C. / Hauer, M. / Zhou, K. / Lin, S. ...Jinek, M. / Jiang, F. / Taylor, D.W. / Sternberg, S.H. / Kaya, E. / Ma, E. / Anders, C. / Hauer, M. / Zhou, K. / Lin, S. / Kaplan, M. / Iavarone, A.T. / Charpentier, E. / Nogales, E. / Doudna, J.A.
CitationJournal: Science / Year: 2014
Title: Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.
Authors: Martin Jinek / Fuguo Jiang / David W Taylor / Samuel H Sternberg / Emine Kaya / Enbo Ma / Carolin Anders / Michael Hauer / Kaihong Zhou / Steven Lin / Matias Kaplan / Anthony T Iavarone / ...Authors: Martin Jinek / Fuguo Jiang / David W Taylor / Samuel H Sternberg / Emine Kaya / Enbo Ma / Carolin Anders / Michael Hauer / Kaihong Zhou / Steven Lin / Matias Kaplan / Anthony T Iavarone / Emmanuelle Charpentier / Eva Nogales / Jennifer A Doudna /
Abstract: Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA ...Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.
History
DepositionJan 16, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2014Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2014Group: Atomic model / Database references
Revision 1.2Mar 26, 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_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.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 / _struct_conn.pdbx_dist_value / _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_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: CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
B: CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)318,4778
Polymers317,9722
Non-polymers5056
Water3,657203
1
A: CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,1783
Polymers158,9861
Non-polymers1922
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,2995
Polymers158,9861
Non-polymers3124
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)159.780, 209.620, 91.260
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212
Noncrystallographic symmetry (NCS)NCS oper: (Code: given
Matrix: (0.99975, -0.02197, 0.0043), (-0.0221, -0.99927, 0.03109), (0.00361, -0.03117, -0.99951)
Vector: -1.20034, -157.83882, 41.34232)

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Components

#1: Protein CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1 / CAS9


Mass: 158986.109 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) STREPTOCOCCUS PYOGENES (bacteria) / Strain: SEROTYPE M1 / Plasmid: PEC-K-MBP / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): ROSETTA2
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 203 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsM-TERMINAL GAAS IS DERIVED FROM THE EXPRESSION VECTOR

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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: 2.41 Å3/Da / Density % sol: 49 % / Description: NONE
Crystal growpH: 8.5
Details: 0.1 M TRIS PH 8.5, 0.3 M LITHIUM SULFATE, 15% PEG 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 31, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.62→47.52 Å / Num. obs: 92408 / % possible obs: 98.2 % / Observed criterion σ(I): 1.94 / Redundancy: 2.3 % / Biso Wilson estimate: 64.77 Å2 / Rmerge(I) obs: 0.05 / Net I/σ(I): 13.02
Reflection shellResolution: 2.62→2.69 Å / Redundancy: 2.3 % / Rmerge(I) obs: 0.64 / Mean I/σ(I) obs: 1.94 / % possible all: 98.5

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE)refinement
XDSdata reduction
XSCALEdata scaling
autoSHARPphasing
RefinementMethod to determine structure: MIRAS
Starting model: NONE

Resolution: 2.62→47.481 Å / SU ML: 0.41 / σ(F): 1.36 / Phase error: 31.27 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2858 2424 2.6 %
Rwork0.2523 --
obs0.2532 92408 99.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.62→47.481 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms18862 0 26 203 19091
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00519183
X-RAY DIFFRACTIONf_angle_d0.94825770
X-RAY DIFFRACTIONf_dihedral_angle_d16.9587339
X-RAY DIFFRACTIONf_chiral_restr0.0532899
X-RAY DIFFRACTIONf_plane_restr0.0033268
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6199-2.67340.34631410.31245216X-RAY DIFFRACTION100
2.6734-2.73150.3481400.29675212X-RAY DIFFRACTION100
2.7315-2.7950.33141420.29215261X-RAY DIFFRACTION100
2.795-2.86490.3181140.30035312X-RAY DIFFRACTION100
2.8649-2.94240.43381970.31955172X-RAY DIFFRACTION100
2.9424-3.02890.33921030.31735306X-RAY DIFFRACTION100
3.0289-3.12670.39041630.30445238X-RAY DIFFRACTION100
3.1267-3.23840.35311370.28915276X-RAY DIFFRACTION100
3.2384-3.3680.29151050.27775315X-RAY DIFFRACTION100
3.368-3.52130.29861950.26365245X-RAY DIFFRACTION100
3.5213-3.70690.29991000.25085298X-RAY DIFFRACTION100
3.7069-3.9390.26721980.24195237X-RAY DIFFRACTION99
3.939-4.2430.2726980.2285321X-RAY DIFFRACTION99
4.243-4.66960.28781930.21295270X-RAY DIFFRACTION99
4.6696-5.34450.25981050.22285395X-RAY DIFFRACTION99
5.3445-6.73050.2739990.26835402X-RAY DIFFRACTION99
6.7305-47.4890.22591940.2355508X-RAY DIFFRACTION98
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)
11.9611-0.3317-0.55771.3429-0.29983.11310.0860.1160.02920.00290.04880.0601-0.5508-0.3253-0.03540.30440.1514-0.04320.21460.00040.0788-36.405-29.7107-24.0474
20.8415-0.05590.46351.0919-0.51671.2968-0.10310.01340.0044-0.01530.0531-0.1155-0.25010.14190.01050.26-0.03110.00130.15590.0290.2398-18.4746-58.94593.974
30.0752-0.0494-0.00880.33910.31760.41710.0434-0.17170.11360.2927-0.188-0.1308-1.01570.9933-0.3391.118-1.0348-0.18540.38890.59340.4699-3.4501-16.0601-13.8044
42.35510.53950.44311.3934-0.01222.12170.1625-0.23880.12220.3479-0.15660.13350.221-0.06730.02220.2543-0.11630.12770.26-0.03470.1256-39.0221-125.311569.1741
50.4942-0.1358-0.16072.1738-0.47190.7753-0.09530.0437-0.0552-0.04240.0421-0.04560.04090.0790.03190.09590.0033-0.01490.22410.070.3003-19.403-96.934540.4661
60.8212-0.0362-0.19330.76230.16480.98680.0013-0.7989-0.8501-0.16590.18530.14710.4582-0.1772-0.02360.4324-0.0616-0.09840.70220.17680.741-4.603-140.119658.6578
70.8727-0.02010.33880.4085-0.03851.8746-0.2565-0.214-0.18350.03950.33750.3252-0.3051-1.3947-0.19690.35710.16350.02910.7350.4250.634-56.0746-83.9324-2.4003
80.98020.1150.10821.16060.66953.1914-0.240.12180.4269-0.00170.02550.8302-0.0196-1.2544-1.30530.17360.129-0.05880.46260.58710.9453-58.0188-71.426946.3373
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1CHAIN A AND ((RESID 4 THROUGH 65) OR (RESID 718 THROUGH 764) OR (RESID 908 THROUGH 1363))
2X-RAY DIFFRACTION2CHAIN A AND (RESID 66 THROUGH 447 )
3X-RAY DIFFRACTION3CHAIN A AND (RESID 775 THROUGH 902 )
4X-RAY DIFFRACTION4CHAIN B AND ((RESID 4 THROUGH 65) OR (RESID 718 THROUGH 764) OR (RESID 908 THROUGH 1363))
5X-RAY DIFFRACTION5CHAIN B AND (RESID 66 THROUGH 447 )
6X-RAY DIFFRACTION6CHAIN B AND (RESID 773 THROUGH 901 )
7X-RAY DIFFRACTION7CHAIN A AND (RESID 503 THROUGH 714)
8X-RAY DIFFRACTION8CHAIN B AND (RESID 502 THROUGH 713)

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