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- PDB-5fw1: Crystal structure of SpyCas9 variant VQR bound to sgRNA and TGAG ... -

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Basic information

Entry
Database: PDB / ID: 5fw1
TitleCrystal structure of SpyCas9 variant VQR bound to sgRNA and TGAG PAM target DNA
Components
  • CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
  • NON-TARGET DNA STRAND
  • SGRNA
  • TARGET DNA STRAND
KeywordsHYDROLASE/DNA / HYDROLASE-DNA COMPLEX / CRISPR / CAS9 / ENDONUCLEASE / PAM / GENOME EDITING / RNP / PROTEIN-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 (bacteria)
SYNTHETIC CONSTRUCT (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.499 Å
AuthorsAnders, C. / Bargsten, K. / Jinek, M.
CitationJournal: Mol.Cell / Year: 2016
Title: Structural Plasticity of Pam Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.
Authors: Anders, C. / Bargsten, K. / Jinek, M.
History
DepositionFeb 11, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 20, 2016Provider: repository / Type: Initial release
Revision 2.0Oct 23, 2019Group: Atomic model / Data collection / Other / Category: atom_site / pdbx_database_status
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _pdbx_database_status.status_code_sf
Revision 2.1Jan 10, 2024Group: 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 / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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.ptnr1_symmetry / _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 / _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: SGRNA
B: CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
C: TARGET DNA STRAND
D: NON-TARGET DNA STRAND
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,51019
Polymers197,9534
Non-polymers55715
Water5,423301
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area22930 Å2
ΔGint-184.4 kcal/mol
Surface area73850 Å2
MethodPISA
Unit cell
Length a, b, c (Å)176.637, 66.984, 187.228
Angle α, β, γ (deg.)90.00, 111.12, 90.00
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 STRAND


Mass: 8543.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#4: DNA chain NON-TARGET DNA STRAND


Mass: 3734.479 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

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RNA chain / Protein , 2 types, 2 molecules AB

#1: RNA chain SGRNA


Mass: 26788.984 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#2: Protein CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1 / SPYCAS9


Mass: 158886.078 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) STREPTOCOCCUS PYOGENES (bacteria) / Strain: M1 / Plasmid: PEC-K-MBP / Production host: ESCHERICHIA COLI (E. coli) / Variant (production host): ROSETTA 2
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

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Non-polymers , 3 types, 316 molecules

#5: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: K
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 301 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsGAAS AT THE N-TERMINUS IS DERIVED FROM EXPRESSION VECTOR. D10A AND H840A MUTATIONS HAVE BEEN ...GAAS AT THE N-TERMINUS IS DERIVED FROM EXPRESSION VECTOR. D10A AND H840A MUTATIONS HAVE BEEN ENGINEERED TO INACTIVATE NUCLEASE DOMAINS. D1135V, R1335Q, T1337R MUTATIONS ARE SPECIFIC TO THE VQR SPYCAS9 VARIANT.

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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: 2.6 Å3/Da / Density % sol: 52 % / Description: NONE
Crystal growpH: 8.5 / Details: 0.1 M TRIS PH 8.5, 0.3 M KSCN, 15% (W/V) PEG 3400

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1
DetectorType: DECTRIS PILATUS / Detector: PIXEL / Date: Oct 22, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.5→47.62 Å / Num. obs: 71152 / % possible obs: 99.4 % / Observed criterion σ(I): 1.7 / Redundancy: 3.5 % / Rmerge(I) obs: 0.1 / Net I/σ(I): 11.9
Reflection shellResolution: 2.5→2.65 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.77 / Mean I/σ(I) obs: 1.7 / % possible all: 98.2

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4UN3
Resolution: 2.499→47.622 Å / SU ML: 0.42 / σ(F): 1.21 / Phase error: 28.09 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2544 6915 5 %
Rwork0.2183 --
obs0.2202 71146 99.51 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.499→47.622 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10672 2526 15 301 13514
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00713714
X-RAY DIFFRACTIONf_angle_d0.78718973
X-RAY DIFFRACTIONf_dihedral_angle_d16.4788029
X-RAY DIFFRACTIONf_chiral_restr0.0452169
X-RAY DIFFRACTIONf_plane_restr0.0041983
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4988-2.52720.44942140.37714009X-RAY DIFFRACTION92
2.5272-2.55690.36322300.31744441X-RAY DIFFRACTION100
2.5569-2.58810.39072420.32774368X-RAY DIFFRACTION100
2.5881-2.62090.3832320.31664363X-RAY DIFFRACTION100
2.6209-2.65540.31452270.28844347X-RAY DIFFRACTION100
2.6554-2.69170.36462200.29444478X-RAY DIFFRACTION100
2.6917-2.73020.38862250.29954302X-RAY DIFFRACTION100
2.7302-2.77090.35352260.2874483X-RAY DIFFRACTION100
2.7709-2.81420.33382500.2774327X-RAY DIFFRACTION100
2.8142-2.86030.32172350.27594382X-RAY DIFFRACTION100
2.8603-2.90970.28592290.26914420X-RAY DIFFRACTION100
2.9097-2.96260.33132240.27444380X-RAY DIFFRACTION100
2.9626-3.01950.31552300.28574351X-RAY DIFFRACTION100
3.0195-3.08120.30582280.27164348X-RAY DIFFRACTION100
3.0812-3.14810.31132380.2774438X-RAY DIFFRACTION100
3.1481-3.22140.29372230.24884354X-RAY DIFFRACTION100
3.2214-3.30190.24492390.24084356X-RAY DIFFRACTION100
3.3019-3.39120.30352370.22924392X-RAY DIFFRACTION100
3.3912-3.49090.24362310.22154429X-RAY DIFFRACTION100
3.4909-3.60360.26192370.21034328X-RAY DIFFRACTION100
3.6036-3.73230.24692290.19654386X-RAY DIFFRACTION100
3.7323-3.88170.19652240.19144338X-RAY DIFFRACTION100
3.8817-4.05820.23112380.18984408X-RAY DIFFRACTION100
4.0582-4.27210.20312250.17754381X-RAY DIFFRACTION100
4.2721-4.53950.19472280.17424387X-RAY DIFFRACTION100
4.5395-4.88970.21612320.1714370X-RAY DIFFRACTION100
4.8897-5.38120.23122270.17334372X-RAY DIFFRACTION100
5.3812-6.15840.26262320.194414X-RAY DIFFRACTION100
6.1584-7.75360.20252310.1974350X-RAY DIFFRACTION100
7.7536-47.6310.1892320.18334408X-RAY DIFFRACTION100
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)
10.3287-0.0022-0.08970.0945-0.06680.40790.06770.0646-0.00070.0643-0.0523-0.0134-0.01410.133-00.2445-0.0507-0.00890.25780.00620.2438185.26163.467639.5485
20.15530.1920.03570.2466-0.1620.48040.07580.38770.19930.4358-0.03090.2769-0.9681-0.8640.0070.71450.17780.07470.6355-0.0180.5033200.282560.855164.4039
30.3506-0.1928-0.2510.08220.1711.07260.03150.0074-0.05580.0799-0.01220.01080.00990.153600.2876-0.0239-0.03570.2830.00210.3575188.335865.880947.7925
40.44830.22660.27150.2928-0.09770.4393-0.09260.0243-0.03370.09360.0461-0.0378-0.15010.0639-00.34410.02390.00150.2711-0.00870.3545171.730477.252675.3028
50.3924-0.64720.27510.2892-0.2190.4179-0.0118-0.0162-0.01580.09480.00460.06110.02660.076-00.3655-0.0318-0.01630.37260.00630.3996165.852947.131266.3036
60.56280.1456-0.07790.31050.0090.2905-0.04840.1011-0.06590.02420.0181-0.00170.1912-0.104800.3421-0.07970.0140.2943-0.0240.3177147.523545.993639.5608
70.5054-0.150.13220.2085-0.07030.43950.09610.09370.0275-0.05820.01190.02120.06270.077800.3328-0.0148-0.0320.318-0.03350.3248179.340762.192331.984
80.3229-0.3456-0.85050.45320.85342.29560.2988-0.3880.8718-0.25460.39-1.1883-0.94981.32260.1080.8788-0.03190.27620.84680.11171.1382138.622774.431436.2429
90.1398-0.0707-0.11650.1846-0.04630.10780.10130.2517-0.31210.0289-0.0118-0.1329-0.0352-0.3296-0.00030.3585-0.0067-0.03860.4224-0.11080.418167.550243.282124.9099
100.1042-0.09730.07020.0045-0.01780.00840.1597-0.12540.23350.02640.05650.01640.13440.11520.00040.394-0.0440.00690.3839-0.00290.3592169.292164.306165.2458
110.26940.042-0.04030.2871-0.08470.02270.14830.1135-0.77470.13520.2386-0.68530.2940.26220.00060.4427-0.0621-0.00340.4117-0.12970.5781165.61244.419725.8528
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1CHAIN 'B' AND (RESID 4 THROUGH 236 )
2X-RAY DIFFRACTION2CHAIN 'B' AND (RESID 237 THROUGH 315 )
3X-RAY DIFFRACTION3CHAIN 'B' AND (RESID 316 THROUGH 551 )
4X-RAY DIFFRACTION4CHAIN 'B' AND (RESID 552 THROUGH 750 )
5X-RAY DIFFRACTION5CHAIN 'B' AND (RESID 751 THROUGH 953 )
6X-RAY DIFFRACTION6CHAIN 'B' AND (RESID 954 THROUGH 1364 )
7X-RAY DIFFRACTION7CHAIN 'A' AND (RESID 1 THROUGH 70 )
8X-RAY DIFFRACTION8CHAIN 'A' AND (RESID 71 THROUGH 81 )
9X-RAY DIFFRACTION9CHAIN 'C' AND (RESID 1 THROUGH 10 )
10X-RAY DIFFRACTION10CHAIN 'C' AND (RESID 11 THROUGH 28 )
11X-RAY DIFFRACTION11CHAIN 'D' AND (RESID 2 THROUGH 12 )

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