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- PDB-7ox7: Target-bound SpCas9 complex with TRAC chimeric RNA-DNA guide -

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Basic information

Entry
Database: PDB / ID: 7ox7
TitleTarget-bound SpCas9 complex with TRAC chimeric RNA-DNA guide
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • TRAC non-target DNA strand
  • TRAC target DNA strand
  • chimeric RNA-DNA guide
KeywordsHYDROLASE / Cas9 / CRISPR / chRDNA / TRAC / nuclease
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) / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsDonohoue, P. / Pacesa, M. / Lau, E. / Vidal, B. / Irby, M.J. / Nyer, D.B. / Rotstein, T. / Banh, L. / Toh, M.T. / Gibson, J. ...Donohoue, P. / Pacesa, M. / Lau, E. / Vidal, B. / Irby, M.J. / Nyer, D.B. / Rotstein, T. / Banh, L. / Toh, M.T. / Gibson, J. / Kohrs, B. / Baek, K. / Owen, A.L.G. / Slorach, E.M. / van Overbeek, M. / Fuller, C.K. / May, A.P. / Jinek, M. / Cameron, P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567 Switzerland
CitationJournal: Mol.Cell / Year: 2021
Title: Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.
Authors: Donohoue, P.D. / Pacesa, M. / Lau, E. / Vidal, B. / Irby, M.J. / Nyer, D.B. / Rotstein, T. / Banh, L. / Toh, M.S. / Gibson, J. / Kohrs, B. / Baek, K. / Owen, A.L.G. / Slorach, E.M. / van ...Authors: Donohoue, P.D. / Pacesa, M. / Lau, E. / Vidal, B. / Irby, M.J. / Nyer, D.B. / Rotstein, T. / Banh, L. / Toh, M.S. / Gibson, J. / Kohrs, B. / Baek, K. / Owen, A.L.G. / Slorach, E.M. / van Overbeek, M. / Fuller, C.K. / May, A.P. / Jinek, M. / Cameron, P.
History
DepositionJun 22, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 15, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: chimeric RNA-DNA guide
B: CRISPR-associated endonuclease Cas9/Csn1
C: TRAC target DNA strand
D: TRAC non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,71920
Polymers197,1234
Non-polymers59616
Water6,918384
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23510 Å2
ΔGint-174 kcal/mol
Surface area75430 Å2
MethodPISA
Unit cell
Length a, b, c (Å)177.542, 67.406, 188.136
Angle α, β, γ (deg.)90.000, 112.542, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain TRAC target DNA strand


Mass: 8574.544 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain TRAC non-target DNA strand


Mass: 3677.415 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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

#1: DNA/RNA hybrid chimeric RNA-DNA guide


Mass: 25995.623 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158875.031 Da / Num. of mol.: 1 / Mutation: D10A, H840A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Plasmid: pMJ841 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2(DE3)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

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Non-polymers , 3 types, 400 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: K
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 384 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: 2.64 Å3/Da / Density % sol: 53.33 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 0.1 M Tris-acetate pH 8.5, 0.3-0.5 M KSCN, 17-19% PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Oct 8, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.6→42.46 Å / Num. obs: 63691 / % possible obs: 99.86 % / Redundancy: 6.8 % / Biso Wilson estimate: 56.09 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.2193 / Net I/σ(I): 8.54
Reflection shellResolution: 2.6→2.693 Å / Redundancy: 6.8 % / Rmerge(I) obs: 2.461 / Mean I/σ(I) obs: 0.74 / Num. unique obs: 6337 / CC1/2: 0.284 / % possible all: 99.83

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Processing

Software
NameVersionClassification
Cootmodel building
PHENIX1.15.2_3472refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5FQ5
Resolution: 2.6→42.46 Å / SU ML: 0.3831 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 26.0583
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.229 3179 4.99 %
Rwork0.209 60474 -
obs0.21 63653 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 79 Å2
Refinement stepCycle: LAST / Resolution: 2.6→42.46 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10831 2498 16 384 13729
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.003413819
X-RAY DIFFRACTIONf_angle_d0.621919146
X-RAY DIFFRACTIONf_chiral_restr0.04072179
X-RAY DIFFRACTIONf_plane_restr0.00382013
X-RAY DIFFRACTIONf_dihedral_angle_d17.04868100
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6-2.640.37271420.35712597X-RAY DIFFRACTION99.71
2.64-2.680.36311220.35542613X-RAY DIFFRACTION99.93
2.68-2.720.37081420.33922606X-RAY DIFFRACTION100
2.72-2.770.33971330.33012596X-RAY DIFFRACTION99.89
2.77-2.820.30531450.31312634X-RAY DIFFRACTION100
2.82-2.880.31121390.30862587X-RAY DIFFRACTION99.82
2.88-2.930.34281410.30852636X-RAY DIFFRACTION99.86
2.93-30.30881220.29632567X-RAY DIFFRACTION99.89
3-3.070.34211510.30472676X-RAY DIFFRACTION99.96
3.07-3.140.32051370.27812570X-RAY DIFFRACTION99.96
3.14-3.230.26941270.25622644X-RAY DIFFRACTION99.89
3.23-3.320.23761480.23262567X-RAY DIFFRACTION99.93
3.32-3.430.23111370.22122633X-RAY DIFFRACTION100
3.43-3.550.27971360.21752661X-RAY DIFFRACTION99.93
3.55-3.70.24151380.20532602X-RAY DIFFRACTION99.89
3.7-3.860.20561350.19112624X-RAY DIFFRACTION99.93
3.86-4.070.1851430.17792636X-RAY DIFFRACTION99.89
4.07-4.320.20611400.17422655X-RAY DIFFRACTION100
4.32-4.660.2071340.16332630X-RAY DIFFRACTION100
4.66-5.120.19011380.15892652X-RAY DIFFRACTION100
5.12-5.860.19231430.16882667X-RAY DIFFRACTION99.96
5.86-7.380.19151410.1832683X-RAY DIFFRACTION99.75
7.38-40.550.15621450.15632738X-RAY DIFFRACTION99.45
Refinement TLS params.

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

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
14.15948026116-0.8069908174490.7736933467621.8275321656-0.09863494638451.483172352770.6760476035150.927487684741-0.7541321373430.1106442594260.125990090665-0.5178457977161.18321056470.4326810797080.399256454528-0.0001981331382030.008277201387670.1081674719980.29529698303-0.109730067480.0296560733891-12.52557208546.0304227104371.1376318942
20.3497371655410.2827804094370.5440694929270.411151939924-0.3051214999050.3619337739150.1285521224090.1721121097840.0294599866558-0.0308007796528-0.03704308799430.03805405301650.1225632866280.04621189350570.4103517950330.351674662313-0.00621227932772-0.06607312452470.361436662487-0.04329792314520.3451862923975.124388829513.0681986287125.7128186422
30.005264382368920.005942008020240.02607116343770.0417586370516-0.0178484260525-0.02342752509960.133921844097-0.1139830264890.479419998305-0.3964043647490.3354059761520.13587558221-0.3251978693310.3386137976893.90681575445E-60.910837216127-0.2317344914350.2468001126561.178310455510.1322202850821.18129453775-38.846410906816.609985051434.4116591808
40.2544493083820.0330959143766-0.00163600020359-0.0526163029979-0.1827773530110.5477429678860.02415755428530.1170504974540.01282483001090.0237343508423-0.03376209005690.030009275960.001148123623450.113642213419-0.0009119715575240.307828633991-0.05062970195750.01023139698640.320096090889-0.03099256964790.374915038178-1.074946857017.5848527070831.2771465133
50.264812500570.09696958149790.416126976919-0.203280594864-0.4825475892160.273711318105-0.17293581690.1058815019790.09508672175420.256645098626-0.06764713816560.130078536264-0.4162282793730.298281392973-0.02342340339540.6464508070130.03169412697690.07368791820880.475649552102-0.012934039930.38411328058128.28296095910.20041503697964.0843103821
60.846091391207-0.249202342065-0.3073800676470.1277827097740.1176199593590.6987466947330.00116426808089-0.132993988345-0.02756778724850.0912852143261-0.01003813947850.086856585154-0.01223782575960.2905349110693.91026761167E-50.326463356244-0.0328552360711-0.02135526128120.3709517603830.02407490269690.32679051835916.45076959475.3740672389639.6832945226
70.3986366435780.1637288279080.1783520551780.6894736536750.1708209700450.831788366155-0.0311834390152-0.06989029285950.04639793001680.2016270016010.1130415401-0.0822719012785-0.1889407615070.1215180212384.48280668638E-50.431739514395-0.00672737579737-0.006397459923550.267262468461-0.03261536377390.336697481799-2.4806313918818.783998282482.8013466728
80.3666540163970.3042603239820.3642102604570.722217598769-0.06463253845910.317229796390.115295069539-0.147001850129-0.2900381367670.09079385503810.0159347965681-0.4769907736770.2834610751690.081944645746-0.0009885317372910.4960530857690.057487941893-0.06967708657880.57680791444-0.02740596167840.77404947191-10.6981815933-8.6258753497664.9954478312
90.9850921235470.13525678155-0.307819336590.4153978193630.05111589535110.841035451583-0.07079584235790.0730275275129-0.07379505934760.05084559187440.0798435634039-0.007477171973420.293512810914-0.0248034468097-0.0004411611644080.369219139777-0.0813397883734-0.0240409634850.249310492701-0.03983243466630.334686355608-29.6446045667-11.401415221843.1465011334
100.0346324400895-0.002343428272950.009535469868140.00167906491687-0.02531191222010.005456860336040.04327548801690.134150742524-0.1495698711550.0903431000629-0.04234176458540.0337626868411-0.017573034473-0.2103787859120.01076854596821.28047006455-0.589218834338-0.05294840428811.56508179571-0.3526993624440.933256737815-15.9150486952-20.886683536918.9873837887
110.02515587746640.007742160109290.004776420318990.00220057722024-0.03116098192330.02756307669030.0145548285598-0.122994501184-0.317842911913-0.0633661234071-0.02308376350770.008139264975980.3370807284150.0315227859702-5.92987488976E-50.488807224888-0.08667847155160.00468971173660.5741022926-0.07560667051160.589015391142-2.58601173911-9.1303061419631.0662570293
12-0.0198035674481-0.00761483859797-0.0405484691413-0.0106911788730.059160284986-0.08619067724150.0699865232575-0.04879025095690.2962395684230.0477184222340.3165448880610.03712552150420.2818264218580.333101749370.0001544838342610.516233639085-0.1276984402030.0476510671520.488461941456-0.06887655990650.486896792365-1.229339590557.0877221526154.9807126707
13-0.006063292456540.004379807608090.0178430544593-0.00618163295325-0.002954668339980.00243721919686-0.0335966451911-0.1244242540840.2505048935850.4052283276120.08424464761630.170113382650.193536342313-0.07995400207570.0002489128896561.2441921903-0.118117167440.2010381312540.6155033311540.003123152571780.819308413529-14.0943960996-0.93202279340374.5366545023
140.0996403617612-0.06036422007140.02659868188030.0483784338259-0.006699398102080.04621432088030.0692960639629-0.00435628036384-0.04534882614060.0292141824196-0.1236200224910.007251781152230.231192508728-0.142263883552-0.006627963732750.839653836166-0.04798729567910.00986240868780.576961877808-0.09042284751330.701163659964-22.11881588619.8188586623682.3581883071
150.4808624025220.0215097123615-0.06108934247310.225928823448-0.0424587963747-0.0139956065849-0.007078305650750.193729683517-0.886731881380.09525811653550.4357374796170.1948326729960.7661599947550.350168868780.02148519280810.594509527277-0.0709385767671-0.01537854790150.471581876004-0.2115366725560.50695356343-11.5507142271-14.059309042823.7856529579
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 10 )
2X-RAY DIFFRACTION2chain 'A' and (resid 11 through 70 )
3X-RAY DIFFRACTION3chain 'A' and (resid 71 through 81 )
4X-RAY DIFFRACTION4chain 'B' and (resid 3 through 163 )
5X-RAY DIFFRACTION5chain 'B' and (resid 164 through 300 )
6X-RAY DIFFRACTION6chain 'B' and (resid 301 through 512 )
7X-RAY DIFFRACTION7chain 'B' and (resid 513 through 692 )
8X-RAY DIFFRACTION8chain 'B' and (resid 693 through 909 )
9X-RAY DIFFRACTION9chain 'B' and (resid 910 through 1366 )
10X-RAY DIFFRACTION10chain 'C' and (resid -7 through -3 )
11X-RAY DIFFRACTION11chain 'C' and (resid -2 through 2 )
12X-RAY DIFFRACTION12chain 'C' and (resid 3 through 12 )
13X-RAY DIFFRACTION13chain 'C' and (resid 13 through 17 )
14X-RAY DIFFRACTION14chain 'C' and (resid 18 through 20 )
15X-RAY DIFFRACTION15chain 'D' and (resid -3 through 7 )

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