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- PDB-5b2p: Crystal structure of Francisella novicida Cas9 in complex with sg... -

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Basic information

Entry
Database: PDB / ID: 5b2p
TitleCrystal structure of Francisella novicida Cas9 in complex with sgRNA and target DNA (TGA PAM)
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (5'-D(*TP*GP*AP*TP*AP*TP*CP*GP*G)-3')
  • Guide RNA
  • Target DNA
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas9 / genome engineering / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR system subtype II-B RNA-guided endonuclease Cas9/Csx12 / : / : / CRISPR-associated endonuclease Cas9 alpha-helical lobe / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9-type HNH domain / Cas9-type HNH domain profile.
Similarity search - Domain/homology
ACETATE ION / DNA / DNA (> 10) / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida U112 (bacteria)
synthetic construct (others)
Francisella tularensis subsp. novicida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.7 Å
AuthorsHirano, H. / Nishimasu, H. / Nakane, T. / Ishitani, R. / Nureki, O.
CitationJournal: Cell / Year: 2016
Title: Structure and Engineering of Francisella novicida Cas9
Authors: Hirano, H. / Gootenberg, J.S. / Horii, T. / Abudayyeh, O.O. / Kimura, M. / Hsu, P.D. / Nakane, T. / Ishitani, R. / Hatada, I. / Zhang, F. / Nishimasu, H. / Nureki, O.
History
DepositionFeb 1, 2016Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 2, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 9, 2016Group: Database references
Revision 1.2Oct 4, 2017Group: Advisory / Data collection / Derived calculations
Category: diffrn_detector / diffrn_source ...diffrn_detector / diffrn_source / pdbx_struct_oper_list / pdbx_unobs_or_zero_occ_atoms
Item: _diffrn_detector.detector / _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 / struct_conn_type
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_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_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.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_ptnr1_label_alt_id / _struct_conn.pdbx_value_order / _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_conn_type.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: Guide RNA
C: Target DNA
D: DNA (5'-D(*TP*GP*AP*TP*AP*TP*CP*GP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)235,53253
Polymers233,0774
Non-polymers2,45549
Water18,9701053
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area32400 Å2
ΔGint-324 kcal/mol
Surface area77280 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.611, 159.273, 96.674
Angle α, β, γ (deg.)90.000, 106.860, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain Target DNA


Mass: 9008.811 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: chemical synthesis
Source: (synth.) Francisella tularensis subsp. novicida (bacteria)
#4: DNA chain DNA (5'-D(*TP*GP*AP*TP*AP*TP*CP*GP*G)-3')


Mass: 2770.834 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida (bacteria)

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Protein / DNA/RNA hybrid , 2 types, 2 molecules AB

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 190995.422 Da / Num. of mol.: 1 / Mutation: N995A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. novicida U112 (bacteria)
Strain: U112 / Gene: cas9, FTN_0757 / Plasmid: pE-SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) Rosetta2
References: UniProt: A0Q5Y3, Hydrolases; Acting on ester bonds
#2: DNA/RNA hybrid Guide RNA


Mass: 30301.904 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: in vitro transcription / Source: (synth.) synthetic construct (others)

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Non-polymers , 7 types, 1102 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na
#7: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#8: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 17 / Source method: obtained synthetically / Formula: Ca
#9: Chemical...
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 23 / Source method: obtained synthetically / Formula: C2H6O2
#10: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1053 / 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: 2.58 Å3/Da / Density % sol: 52.25 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5
Details: 9-11% PEG 3350, 0.2 M calcium acetate, 0.1 M sodium acetate

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 4, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.7→46.26 Å / Num. obs: 255229 / % possible obs: 98.8 % / Redundancy: 14.1 % / Net I/σ(I): 19.6

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Processing

Software
NameVersionClassification
PHENIX1.10_2155: ???refinement
Aimlessdata scaling
PDB_EXTRACT3.2data extraction
RefinementMethod to determine structure: SAD / Resolution: 1.7→46.259 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 20.66
RfactorNum. reflection% reflection
Rfree0.2032 12766 5 %
Rwork0.1801 --
obs0.1813 255162 98.78 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 143.88 Å2 / Biso mean: 47.3789 Å2 / Biso min: 16.44 Å2
Refinement stepCycle: final / Resolution: 1.7→46.259 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11697 2771 124 1053 15645
Biso mean--46.54 41.37 -
Num. residues----1588
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01415219
X-RAY DIFFRACTIONf_angle_d1.39121133
X-RAY DIFFRACTIONf_chiral_restr0.0872415
X-RAY DIFFRACTIONf_plane_restr0.012233
X-RAY DIFFRACTIONf_dihedral_angle_d16.4898862
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 (%)
1.7-1.71930.31624190.27897895831498
1.7193-1.73960.31854140.27578069848398
1.7396-1.76080.28954240.25697986841098
1.7608-1.78310.26654320.24938006843898
1.7831-1.80650.26384190.23528066848598
1.8065-1.83130.26264300.23037996842698
1.8313-1.85740.26514160.22398084850099
1.8574-1.88520.24634220.2188038846099
1.8852-1.91460.23074270.20798040846799
1.9146-1.9460.23584170.20727947836497
1.946-1.97960.20834190.19028046846599
1.9796-2.01560.22494210.19068084850599
2.0156-2.05430.22264180.19188074849299
2.0543-2.09630.21854310.1878106853799
2.0963-2.14180.2084320.18718089852199
2.1418-2.19170.21214230.18868093851699
2.1917-2.24650.19684330.17618091852499
2.2465-2.30720.20874270.17868118854599
2.3072-2.37510.2094240.17827940836497
2.3751-2.45170.21094250.18038150857599
2.4517-2.53940.21124270.17988148857599
2.5394-2.6410.21524320.182181198551100
2.641-2.76120.22524330.185981498582100
2.7612-2.90680.20144280.183381798607100
2.9068-3.08890.20054200.17957971839197
3.0889-3.32730.19324340.170581998633100
3.3273-3.6620.20074280.172981778605100
3.662-4.19160.18954360.158782128648100
4.1916-5.27980.17254200.15148081850198
5.2798-46.27570.17974350.18748243867899
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)
11.2672-0.18671.20940.5866-0.07354.531-0.0586-0.19210.16170.13360.0671-0.044-0.0837-0.07280.04210.14080.00290.00450.1254-0.05650.19133.98752.385225.1057
21.51881.9979-0.72332.0233-1.0865-0.099-0.07890.2841-1.0829-0.29220.05980.10320.449-0.1040.02910.6502-0.1023-0.01480.3831-0.11471.1728-15.9128-33.82020.0386
31.4835-0.39981.25290.3757-0.18091.80330.0380.31330.050.029-0.0203-0.07350.12130.4902-0.01230.2016-0.00040.0360.2904-0.02430.228627.1952-8.288522.5428
41.00550.03220.42062.52981.18290.848-0.0435-0.075-0.41370.51270.17290.18790.50340.0877-0.09230.52530.04190.03910.3173-0.01910.399325.44-32.066330.3312
51.4052-0.48940.3050.5953-0.90351.960.0022-0.5252-0.40540.16390.19620.06720.5298-0.3972-0.15970.5864-0.03-0.01910.56220.04910.43466.8998-17.193455.0789
62.50510.0654-0.29633.1893-0.59192.4255-0.1096-0.7617-0.30220.38020.03850.02910.2549-0.35590.05140.3247-0.0014-0.01670.5292-0.00210.19369.718-9.18355.9283
71.1437-0.40870.50010.6466-0.27140.8386-0.1295-0.14180.14090.06720.05590.0593-0.2301-0.13570.04990.2412-0.00270.02430.1557-0.02570.2436-20.605612.83612.5188
81.18090.16280.02421.94410.91932.21110.0458-0.49370.22871.0669-0.06790.5376-0.2326-0.14710.06120.85840.08220.14790.4178-0.14480.5005-17.891519.396340.5393
91.4526-0.52680.84040.6448-0.22581.27190.064-0.0702-0.24290.03630.04130.06590.23660.0554-0.15660.2513-0.0160.03040.18470.00540.26241.2824-10.560317.5501
104.9465-1.72950.37635.1633-1.2433.65150.14090.81710.0857-0.782-0.14410.4753-0.331-0.0036-0.04870.42330.0332-0.14750.4293-0.05140.3818-34.06718.3312-16.1103
110.8299-0.69640.08980.6137-0.06870.68810.01770.11310.0235-0.0595-0.04070.0476-0.06750.04460.03750.2287-0.0231-0.00640.17820.01610.2268-15.5436.2892-0.1647
121.632-0.5862-2.37960.23180.83583.47570.2638-0.08850.68410.0388-0.3390.1178-0.91220.35670.07290.4215-0.1443-0.00550.34070.06480.46167.945613.48628.6041
132.5476-0.02420.26491.0371-0.17721.9051-0.1935-0.0730.2207-0.09030.0502-0.05-0.16840.10890.1330.2235-0.0052-0.00750.2044-0.02880.25462.60866.975422.2841
147.28163.4817-0.58085.1149-4.10894.673-0.0136-0.54950.81560.6671-0.029-0.2411-0.6828-0.52890.00560.25090.0851-0.06420.2786-0.07310.23479.72767.929530.1838
154.5365-1.82111.4051.2312-0.39120.5062-0.04040.54490.693-0.0912-0.1695-0.3439-0.06620.36960.20410.2276-0.04770.01570.40620.03840.312625.62910.414715.7656
163.76882.8039-3.7054.0115-3.47863.9144-0.13911.0208-0.677-1.0366-0.1977-0.72421.23980.54320.26720.44960.12740.0710.6154-0.10850.352527.4997-14.53119.0034
173.9144-0.04490.5943.0007-1.36422.6768-0.05530.27070.67830.0508-0.1266-0.2875-0.26440.29840.15150.1886-0.0446-0.00730.3740.02370.365725.3745-0.340216.1072
184.1863-3.3195-1.88183.49620.77743.9169-0.35870.14470.50320.16420.1341.4162-0.6019-1.02870.22040.41780.15310.06150.4852-0.07331.1246-27.505612.798426.6371
191.6891-0.74860.99511.0484-0.19580.9273-0.1258-0.4541-0.4280.41920.21390.17480.1042-0.1254-0.13770.43130.05480.08220.30370.0970.306410.2668-20.534729.1956
206.2978-2.616-1.06681.13980.15692.1912-0.25230.12851.09090.32680.25621.7824-0.7993-1.26850.01070.43490.2192-0.02840.6751-0.00941.2313-28.578216.224925.6487
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 99 )A1 - 99
2X-RAY DIFFRACTION2chain 'A' and (resid 100 through 324 )A100 - 324
3X-RAY DIFFRACTION3chain 'A' and (resid 325 through 643 )A325 - 643
4X-RAY DIFFRACTION4chain 'A' and (resid 644 through 859 )A644 - 859
5X-RAY DIFFRACTION5chain 'A' and (resid 860 through 1065 )A860 - 1065
6X-RAY DIFFRACTION6chain 'A' and (resid 1066 through 1176 )A0
7X-RAY DIFFRACTION7chain 'A' and (resid 1177 through 1496 )A0
8X-RAY DIFFRACTION8chain 'A' and (resid 1497 through 1622 )A0
9X-RAY DIFFRACTION9chain 'B' and (resid 1 through 30 )B1 - 30
10X-RAY DIFFRACTION10chain 'B' and (resid 31 through 40 )B31 - 40
11X-RAY DIFFRACTION11chain 'B' and (resid 41 through 55 )B41 - 55
12X-RAY DIFFRACTION12chain 'B' and (resid 56 through 60 )B56 - 60
13X-RAY DIFFRACTION13chain 'B' and (resid 61 through 65 )B61 - 65
14X-RAY DIFFRACTION14chain 'B' and (resid 66 through 70 )B66 - 70
15X-RAY DIFFRACTION15chain 'B' and (resid 71 through 80 )B71 - 80
16X-RAY DIFFRACTION16chain 'B' and (resid 81 through 85 )B81 - 85
17X-RAY DIFFRACTION17chain 'B' and (resid 86 through 94 )B86 - 94
18X-RAY DIFFRACTION18chain 'C' and (resid 1 through 10 )C1 - 10
19X-RAY DIFFRACTION19chain 'C' and (resid 11 through 30 )C11 - 30
20X-RAY DIFFRACTION20chain 'D' and (resid 1 through 9 )D1 - 9

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