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- PDB-4oo8: Crystal structure of Streptococcus pyogenes Cas9 in complex with ... -

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Basic information

Entry
Database: PDB / ID: 4oo8
TitleCrystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (5'-D(*CP*CP*AP*GP*CP*CP*AP*AP*GP*CP*GP*CP*AP*CP*CP*TP*AP*AP*TP*TP*TP*CP*C)-3')
  • RNA (97-MER)
KeywordsHydrolase/DNA/RNA / Hydrolase-DNA-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 serotype M1 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
AuthorsNishimasu, H. / Ishitani, R. / Nureki, O.
CitationJournal: Cell(Cambridge,Mass.) / Year: 2014
Title: Crystal structure of Cas9 in complex with guide RNA and target DNA
Authors: Nishimasu, H. / Ran, F.A. / Hsu, P.D. / Konermann, S. / Shehata, S.I. / Dohmae, N. / Ishitani, R. / Zhang, F. / Nureki, O.
History
DepositionJan 31, 2014Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Feb 26, 2014Provider: repository / Type: Initial release
Revision 1.1Dec 24, 2014Group: Database references
Revision 1.2Oct 23, 2019Group: Data collection / Database references / Category: reflns_shell / struct_ref_seq_dif
Item: _reflns_shell.Rmerge_I_obs / _struct_ref_seq_dif.details
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.4Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9/Csn1
B: RNA (97-MER)
C: DNA (5'-D(*CP*CP*AP*GP*CP*CP*AP*AP*GP*CP*GP*CP*AP*CP*CP*TP*AP*AP*TP*TP*TP*CP*C)-3')
D: CRISPR-associated endonuclease Cas9/Csn1
E: RNA (97-MER)
F: DNA (5'-D(*CP*CP*AP*GP*CP*CP*AP*AP*GP*CP*GP*CP*AP*CP*CP*TP*AP*AP*TP*TP*TP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)395,1696
Polymers395,1696
Non-polymers00
Water2,594144
1
A: CRISPR-associated endonuclease Cas9/Csn1
B: RNA (97-MER)
C: DNA (5'-D(*CP*CP*AP*GP*CP*CP*AP*AP*GP*CP*GP*CP*AP*CP*CP*TP*AP*AP*TP*TP*TP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)197,5843
Polymers197,5843
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18170 Å2
ΔGint-158 kcal/mol
Surface area72230 Å2
MethodPISA
2
D: CRISPR-associated endonuclease Cas9/Csn1
E: RNA (97-MER)
F: DNA (5'-D(*CP*CP*AP*GP*CP*CP*AP*AP*GP*CP*GP*CP*AP*CP*CP*TP*AP*AP*TP*TP*TP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)197,5843
Polymers197,5843
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17020 Å2
ΔGint-138 kcal/mol
Surface area66610 Å2
MethodPISA
Unit cell
Length a, b, c (Å)76.713, 105.694, 126.817
Angle α, β, γ (deg.)97.68, 98.43, 100.31
Int Tables number1
Space group name H-MP1

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Components

#1: Protein CRISPR-associated endonuclease Cas9/Csn1


Mass: 158964.062 Da / Num. of mol.: 2 / Mutation: D10A/C80L/H840A/C574E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9 / Plasmid: pCold-GST / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2 (DE3)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: RNA chain RNA (97-MER)


Mass: 31689.803 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Single guide RNA
#3: DNA chain DNA (5'-D(*CP*CP*AP*GP*CP*CP*AP*AP*GP*CP*GP*CP*AP*CP*CP*TP*AP*AP*TP*TP*TP*CP*C)-3')


Mass: 6930.489 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Target DNA
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 144 / 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: 2.5 Å3/Da / Density % sol: 50.76 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 12% PEG 3350, 100mM Tris-HCl, 200mM ammonium acetate, 150mM NaCl, 100mM NDSB-256, pH 8.0, vapor diffusion, hanging drop, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL32XU / Wavelength: 1 Å
DetectorType: RAYONIX MX-225 / Detector: CCD / Date: Nov 12, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.5→46.883 Å / Num. obs: 130485 / % possible obs: 98.2 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 59.84 Å2 / Rmerge(I) obs: 0.063 / Χ2: 0.946 / Net I/σ(I): 25.27
Reflection shell

Rmerge(I) obs: 1.1 / Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Highest resolution (Å)Rmerge F obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsRrim(I) all% possible all
2.5-2.650.7952.116272421458206431.13496.2
2.65-2.830.9293.8415743920084197290.61898.2
2.83-3.060.9746.8214687918694183940.33998.4
3.06-3.350.99614.2613557717223169910.15198.7
3.35-3.740.99827.3612253415574153960.07698.9
3.74-4.320.99945.7310783313713135850.04299.1
4.32-5.28162.679154111667115880.0399.3
5.28-7.41167.870273901189590.02799.4
7.41187.0637075508048960.01996.4

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Processing

Software
NameVersionClassificationNB
XSCALEdata scaling
PHENIX1.8.3_1479refinement
PDB_EXTRACT3.14data extraction
BSSdata collection
XDSdata reduction
XDSdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: SeMet-labelled Cas9

Resolution: 2.5→46.701 Å / SU ML: 0.37 / σ(F): 1.98 / Phase error: 29.89 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2536 6521 5 %RANDOM
Rwork0.2219 ---
all0.2235 130396 --
obs0.2235 130396 98.53 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 206.9 Å2 / Biso mean: 80.5122 Å2 / Biso min: 25.17 Å2
Refinement stepCycle: LAST / Resolution: 2.5→46.701 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms18997 5012 0 144 24153
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00224952
X-RAY DIFFRACTIONf_angle_d0.52634943
X-RAY DIFFRACTIONf_chiral_restr0.0224174
X-RAY DIFFRACTIONf_plane_restr0.0023612
X-RAY DIFFRACTIONf_dihedral_angle_d12.589664
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.5-2.52840.37892190.34144151437098
2.5284-2.55820.35752160.32234100431698
2.5582-2.58940.34142160.3184100431698
2.5894-2.62210.35732150.31584091430698
2.6221-2.65660.34382170.31244127434498
2.6566-2.6930.32392150.29984068428398
2.693-2.73150.34682160.29514110432698
2.7315-2.77230.34492170.29124128434598
2.7723-2.81560.32742170.28874125434298
2.8156-2.86170.32032170.27814109432698
2.8617-2.91110.30912160.29324098431498
2.9111-2.9640.32882190.29454169438898
2.964-3.0210.30272170.28774113433098
3.021-3.08260.30692180.27864151436999
3.0826-3.14970.30172150.26664091430699
3.1497-3.22290.30392190.25844156437599
3.2229-3.30350.2872180.24874148436699
3.3035-3.39280.27452150.24624085430099
3.3928-3.49260.26892200.24344182440299
3.4926-3.60530.26522180.23954134435299
3.6053-3.73410.24192180.22044143436199
3.7341-3.88350.25952170.21524132434999
3.8835-4.06020.25522200.20754186440699
4.0602-4.27410.23682190.19744154437399
4.2741-4.54170.23772180.19074132435099
4.5417-4.8920.19942180.18444152437099
4.892-5.38360.23412190.18814165438499
5.3836-6.16120.22532200.199441714391100
6.1612-7.75660.20762200.194941834403100
7.7566-46.70960.18742120.16574021423396
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)
10.6309-0.2809-0.01031.12530.12810.87490.08590.22250.2645-0.0921-0.0323-0.2022-0.11150.0605-0.00060.2224-0.06010.00360.45890.05940.4072-11.6525110.4606-35.8384
20.5319-0.3274-0.0882-0.0175-0.09810.8302-0.05650.15450.0519-0.03010.1337-0.22620.20020.10610.00610.6418-0.10620.0660.47340.0150.3804-14.30980.1578-43.6269
30.3505-0.3568-0.37151.5652-0.3611.9865-0.0850.0745-0.09930.16060.16650.1740.4065-0.57750.10.4713-0.175-0.05990.5230.02710.326-28.299373.9036-12.8286
40.46090.1187-0.52060.0142-0.15420.31810.00010.5302-0.0414-0.0142-0.2497-0.32970.2924-0.212-0.01630.4877-0.00020.120.7169-0.05660.5753-9.043994.9402-36.3115
50.42480.1923-0.44840.1268-0.01660.5382-0.0409-0.03860.31540.63520.1320.1256-0.0271-0.300200.7030.06320.16540.5908-0.01160.5996-39.4881124.5033-8.3969
60.45380.1311-0.09630.1714-0.12620.2027-0.1658-0.12120.07190.699-0.0018-1.3588-0.18490.2495-0.05330.6481-0.0667-0.28890.5731-0.04020.6996-6.995497.9952-8.1476
71.0473-0.3107-0.60780.49630.1781.1496-0.1394-0.2370.02380.0156-0.1338-0.67541.39330.9134-0.06091.33790.0274-0.1910.39450.10510.6365-11.301860.0376-4.2289
80.0545-0.10640.03860.2272-0.23070.1297-0.04190.4497-0.29110.054-0.1483-0.37190.50510.095-00.64260.02150.06220.63830.01480.5276-14.3996103.8534-30.7869
90.0257-0.0064-0.0467-0.0341-0.00160.03060.32370.4978-0.6332-0.1971-0.33220.0727-0.08580.02070.00040.80630.05540.08730.9868-0.26880.8176-4.679380.0954-47.1173
100.56480.33840.22790.76410.28280.56950.0614-0.0591-0.01090.27130.0592-0.18330.1886-0.12410.00510.43110.0391-0.02590.39750.03550.347125.10946.362137.5877
110.4030.3199-0.08391.41240.29350.3506-0.0761-0.17620.21330.2448-0.14560.47580.1688-0.1768-0.00430.527-0.03250.11860.5362-0.06180.50678.98732.468558.8687
121.6587-0.1072-0.0420.31110.12730.942-0.10660.34070.29020.0087-0.0356-0.1203-0.2223-0.1705-00.42080.0058-0.02190.38370.0710.487130.388249.249419.3747
130.07370.33920.32221.22680.35210.4526-0.0682-0.0166-0.05450.22710.1132-0.32590.08260.03750.00470.40270.01170.03130.4973-0.01450.574837.241813.878827.95
140.77080.4198-0.30350.57910.3710.6569-0.1779-0.19481.16060.32380.0602-0.1645-0.28610.2968-0.00170.8533-0.0364-0.20870.6019-0.10841.307740.796564.989431.2626
150.01920.00560.00430.0254-0.00290.0022-0.22590.27290.341-0.30520.1155-0.054-0.43840.1450.00120.6448-0.03780.04730.65990.13750.838531.34218.342420.857
160.00030.00080.00890.0260.0020.0360.07960.322-0.01830.44570.0001-0.1726-0.4092-0.5002-00.54670.05320.08850.62020.01120.628821.161214.034740.7693
170.01310.00270.0060.0219-0.00990.00920.11980.12360.25980.2501-0.33710.16230.0381-0.49710.00010.51960.16490.17740.74280.05910.675515.219231.504144.6599
18-0.00850.00520.00160.002-0.0005-0.00380.2051-0.02550.0478-0.081-0.75590.37020.2894-0.04670.00030.85320.22660.07721.0981-0.14891.2044-1.362739.912847.1558
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1CHAIN A AND (RESID 3 THROUGH 678 )
2X-RAY DIFFRACTION2CHAIN A AND (RESID 679 THROUGH 959 )
3X-RAY DIFFRACTION3CHAIN A AND (RESID 960 THROUGH 1366 )
4X-RAY DIFFRACTION4CHAIN B AND (RESID 1 THROUGH 20 )
5X-RAY DIFFRACTION5CHAIN B AND (RESID 21 THROUGH 52 )
6X-RAY DIFFRACTION6CHAIN B AND (RESID 53 THROUGH 70 )
7X-RAY DIFFRACTION7CHAIN B AND (RESID 71 THROUGH 97 )
8X-RAY DIFFRACTION8CHAIN C AND (RESID 1 THROUGH 10 )
9X-RAY DIFFRACTION9CHAIN C AND (RESID 11 THROUGH 20 )
10X-RAY DIFFRACTION10CHAIN D AND (RESID 3 THROUGH 551 )
11X-RAY DIFFRACTION11CHAIN D AND (RESID 552 THROUGH 919 )
12X-RAY DIFFRACTION12CHAIN D AND (RESID 920 THROUGH 1366 )
13X-RAY DIFFRACTION13CHAIN E AND (RESID 1 THROUGH 70 )
14X-RAY DIFFRACTION14CHAIN E AND (RESID 71 THROUGH 97 )
15X-RAY DIFFRACTION15CHAIN F AND (RESID -1 THROUGH 3 )
16X-RAY DIFFRACTION16CHAIN F AND (RESID 4 THROUGH 8 )
17X-RAY DIFFRACTION17CHAIN F AND (RESID 9 THROUGH 13 )
18X-RAY DIFFRACTION18CHAIN F AND (RESID 14 THROUGH 20 )

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