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- PDB-5b2r: Crystal structure of the Streptococcus pyogenes Cas9 VQR variant ... -

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Basic information

Entry
Database: PDB / ID: 5b2r
TitleCrystal structure of the Streptococcus pyogenes Cas9 VQR variant in complex with sgRNA and target DNA (TGA PAM)
Components
  • CRISPR-associated endonuclease Cas9
  • Guide RNA
  • Non-target DNA, DNA (5'-D(*TP*GP*AP*GP*AP*TP*TP*G)-3')
  • Target DNA
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas9 / genome engineering / HYDROLASE-RNA-DNA 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
ACETATE ION / : / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
Streptococcus pyogenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsHirano, S. / Nishimasu, H. / Ishitani, R. / Nureki, O.
CitationJournal: Mol.Cell / Year: 2016
Title: Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9
Authors: Hirano, S. / Nishimasu, H. / Ishitani, R. / Nureki, O.
History
DepositionFeb 2, 2016Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 23, 2016Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2016Group: Database references
Revision 1.2Oct 4, 2017Group: Data collection / Database references / Derived calculations
Category: citation / diffrn_detector ...citation / diffrn_detector / diffrn_source / pdbx_struct_oper_list
Item: _citation.journal_id_CSD / _diffrn_detector.detector ..._citation.journal_id_CSD / _diffrn_detector.detector / _diffrn_source.pdbx_synchrotron_site / _pdbx_struct_oper_list.symmetry_operation
Revision 1.3Nov 8, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
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.ptnr2_symmetry / _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_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Guide RNA
B: CRISPR-associated endonuclease Cas9
C: Target DNA
D: Non-target DNA, DNA (5'-D(*TP*GP*AP*GP*AP*TP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,93123
Polymers196,1214
Non-polymers81019
Water13,583754
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23310 Å2
ΔGint-172 kcal/mol
Surface area73050 Å2
MethodPISA
Unit cell
Length a, b, c (Å)177.764, 67.744, 187.594
Angle α, β, γ (deg.)90.000, 111.230, 90.000
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


Mass: 8454.472 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: chemical synthesis / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: DNA chain Non-target DNA, DNA (5'-D(*TP*GP*AP*GP*AP*TP*TP*G)-3')


Mass: 2481.652 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)

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RNA chain / Protein , 2 types, 2 molecules AB

#1: RNA chain Guide RNA


Mass: 26209.588 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: in vitro transcription / Source: (synth.) Streptococcus pyogenes (bacteria)
#2: Protein CRISPR-associated endonuclease Cas9 / SpyCas9


Mass: 158975.109 Da / Num. of mol.: 1 / Mutation: D10A, C80L, C574E, H840A, D1135V, R1335Q, T1337R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Strain: serotype M1 / Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

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Non-polymers , 5 types, 773 molecules

#5: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: K
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#7: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#8: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 754 / 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: 2.68 Å3/Da / Density % sol: 54.18 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: 15-17% PEG 3350, 0.4 M KSCN, 0.1 M Tris-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: Oct 16, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2→47.94 Å / Num. obs: 137063 / % possible obs: 97.6 % / Redundancy: 3.5 % / Net I/σ(I): 10.2
Reflection shellResolution: 2→2.03 Å

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Processing

Software
NameVersionClassification
Aimlessdata scaling
PHENIX1.10_2155: ???refinement
PDB_EXTRACT3.2data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4UN3

4un3


Resolution: 2→47.938 Å / SU ML: 0.25 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 25.75
RfactorNum. reflection% reflection
Rfree0.2298 6812 4.97 %
Rwork0.2005 --
obs0.202 136985 97.23 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 210.27 Å2 / Biso mean: 54.6703 Å2 / Biso min: 18.75 Å2
Refinement stepCycle: final / Resolution: 2→47.938 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10539 2464 34 754 13791
Biso mean--54.49 43.27 -
Num. residues----1436
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00513513
X-RAY DIFFRACTIONf_angle_d0.74418767
X-RAY DIFFRACTIONf_chiral_restr0.0442173
X-RAY DIFFRACTIONf_plane_restr0.0041974
X-RAY DIFFRACTIONf_dihedral_angle_d17.5167879
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 (%)
2-2.02270.33962200.31064325454597
2.0227-2.04650.34162430.30624290453397
2.0465-2.07150.32552290.29434307453697
2.0715-2.09770.30322390.27764263450297
2.0977-2.12530.34042390.27054357459697
2.1253-2.15440.28642310.25934260449197
2.1544-2.18520.30492370.26024331456897
2.1852-2.21780.28492210.24684282450397
2.2178-2.25250.26682330.24874057429092
2.2525-2.28940.252260.23114242446895
2.2894-2.32890.27682500.22744355460598
2.3289-2.37120.27722350.22384291452698
2.3712-2.41680.25352020.22574392459498
2.4168-2.46620.28282330.22814318455198
2.4662-2.51980.28012210.22754381460298
2.5198-2.57840.27682220.21724332455498
2.5784-2.64290.24622180.21654390460898
2.6429-2.71430.27172020.21654380458297
2.7143-2.79420.2422250.21154120434594
2.7942-2.88440.24642020.21474413461599
2.8844-2.98740.23912200.21354422464299
2.9874-3.1070.23692320.21174358459098
3.107-3.24840.22252400.20034406464699
3.2484-3.41960.22022260.19064407463398
3.4196-3.63380.21812040.19384214441894
3.6338-3.91420.22862170.17454466468399
3.9142-4.30790.17892270.16594462468999
4.3079-4.93070.16982260.15534440466698
4.9307-6.21010.19052270.17324393462097
6.2101-47.95150.20792650.18714519478497
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)
16.0623-2.1050.74554.63984.54379.0357-0.0001-0.1195-0.40970.1333-0.10360.26060.8322-0.95570.13830.5437-0.10780.09010.4224-0.03940.3952-11.55195.609671.8273
21.1859-0.1097-0.14880.8481-0.15052.59510.07510.2199-0.0671-0.08490.0334-0.09690.56640.4359-0.01380.31480.0227-0.03070.3632-0.06430.269311.4424-0.853924.7262
39.065-1.4838-2.88672.5118-1.32242.43440.23880.8982-0.5843-0.60370.63590.71431.35710.5699-0.88511.26170.1778-0.21181.0438-0.24440.747721.0596-20.01636.0755
40.89430.43780.36270.4131-0.1321.5070.07530.20490.0771-0.02-0.03930.07380.00260.1424-0.03970.2114-0.036-0.01540.225-0.00590.2237-3.40529.471629.5344
52.5169-1.0482-1.18766.6508-1.99085.0226-0.67811.60913.2432-2.59050.0653-0.6766-2.91432.02260.53762.049-0.30640.1481.0330.38561.7501-37.691219.37829.3506
61.9331-0.6703-1.90291.05121.04522.33230.8233-1.23381.0183-0.4483-0.1962-1.4372-2.4110.7742-0.5371.1191-0.28160.17721.0918-0.24621.0136-40.998713.78837.9241
71.54690.3471-0.46170.4033-0.07941.66080.03660.07340.062-0.00280.001-0.0074-0.01930.2969-0.03880.2252-0.0221-0.02210.2003-0.02070.2319-0.94817.167430.9749
82.4293-1.79163.29383.2976-6.1696.6877-0.0086-0.0656-0.27230.47-0.195-0.1047-0.64910.40070.20590.6875-0.06470.00560.5804-0.09190.470827.1843-0.333664.5066
90.4019-0.1601-0.1340.16620.34222.9034-0.0658-0.04370.00110.15870.1139-0.0331-0.05320.3507-0.04060.2862-0.0384-0.01010.2523-0.00650.33677.334312.328660.2912
102.6536-1.68480.62791.5162-0.58531.1363-0.0691-0.1938-0.12980.16410.0912-0.22920.32270.125-0.02530.5625-0.0602-0.02350.4684-0.08610.5217-9.4907-12.350363.6664
111.38280.2106-0.40620.59740.17532.249-0.07250.0486-0.26840.14710.03610.05090.7301-0.38930.02020.4736-0.15820.00330.2592-0.0350.3565-29.7097-11.897342.8526
128.62515.49893.8397.77464.59663.120.07940.5492-1.2818-0.29670.0323-0.2350.3543-0.0095-0.14620.529-0.0295-0.02930.5381-0.13590.5383-9.0431-15.773224.976
132.1301-0.3451-3.05540.51810.73187.9889-0.0435-0.2267-0.04750.15940.10840.21510.4425-0.0499-0.05860.3964-0.06640.00610.41830.02490.3384-7.45885.628165.055
142.7436-2.9946-1.23893.87780.29926.65110.47730.9304-1.0641-0.7693-0.0282-1.19691.2307-0.2408-0.37660.6765-0.09870.06820.4616-0.32650.8063-11.2435-15.63720.5136
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 10 )A1 - 10
2X-RAY DIFFRACTION2chain 'A' and (resid 11 through 35 )A11 - 35
3X-RAY DIFFRACTION3chain 'A' and (resid 36 through 40 )A36 - 40
4X-RAY DIFFRACTION4chain 'A' and (resid 41 through 70 )A41 - 70
5X-RAY DIFFRACTION5chain 'A' and (resid 71 through 75 )A71 - 75
6X-RAY DIFFRACTION6chain 'A' and (resid 76 through 81 )A76 - 81
7X-RAY DIFFRACTION7chain 'B' and (resid 4 through 163 )B4 - 163
8X-RAY DIFFRACTION8chain 'B' and (resid 164 through 299 )B164 - 299
9X-RAY DIFFRACTION9chain 'B' and (resid 300 through 711 )B300 - 711
10X-RAY DIFFRACTION10chain 'B' and (resid 712 through 909 )B712 - 909
11X-RAY DIFFRACTION11chain 'B' and (resid 910 through 1366 )B910 - 1366
12X-RAY DIFFRACTION12chain 'C' and (resid 1 through 10 )C1 - 10
13X-RAY DIFFRACTION13chain 'C' and (resid 11 through 28 )C11 - 28
14X-RAY DIFFRACTION14chain 'D' and (resid 5 through 12 )D5 - 12

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