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- PDB-6i1k: Crystal structure of catalytically inactive FnCas12a in complex w... -

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Basic information

Entry
Database: PDB / ID: 6i1k
TitleCrystal structure of catalytically inactive FnCas12a in complex with a crRNA guide and a dsDNA target
Components
  • CRISPR-associated endonuclease Cas12a
  • DNA non-target strand (33-MER)
  • DNA target strand (38-MER)
  • crRNA (40-MER)
KeywordsHYDROLASE / CRISPR-Cas12a / FnCas12a / CRISPR-Cpf1 / FnCpf1
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
: / CRISPR-associated endonuclease Cpf1 PI domain / : / CRISPR-associated endonuclease Cpf1 REC2 domain / CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.65 Å
AuthorsJinek, M. / Swarts, D.C.
Funding support Germany, Switzerland, 3items
OrganizationGrant numberCountry
European Molecular Biology OrganizationALTF 179-2015 Germany
European Molecular Biology OrganizationaALTF 509-2017 Germany
Swiss National Science FoundationSNSF 31003A_149393 Switzerland
CitationJournal: Mol. Cell / Year: 2019
Title: Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a.
Authors: Swarts, D.C. / Jinek, M.
History
DepositionOct 29, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Data collection / Database references
Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Jan 24, 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
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.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
Revision 1.3Oct 9, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas12a
B: crRNA (40-MER)
C: DNA target strand (38-MER)
D: DNA non-target strand (33-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)189,45729
Polymers188,3214
Non-polymers1,13625
Water1,27971
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area22770 Å2
ΔGint-173 kcal/mol
Surface area73850 Å2
MethodPISA
Unit cell
Length a, b, c (Å)82.172, 141.681, 89.478
Angle α, β, γ (deg.)90.00, 97.43, 90.00
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 DNA target strand (38-MER)


Mass: 11666.548 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: DNA target strand / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA non-target strand (33-MER)


Mass: 11617.511 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: DNA non-target strand (33-MER) / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein CRISPR-associated endonuclease Cas12a / CRISPR-associated endonuclease Cpf1 / FnCas12a / FnCpf1


Mass: 152282.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. novicida (strain U112) (bacteria)
Strain: U112 / Gene: cas12a, cpf1, FTN_1397 / Plasmid: pML-1B / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0Q7Q2, deoxyribonuclease I, EC: 3.1.27.2
#2: RNA chain crRNA (40-MER)


Mass: 12754.537 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: CRISPR RNA (crRNA) guide / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 96 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Mg
#6: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C2H6O2
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 71 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationY

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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: 2.74 Å3/Da / Density % sol: 55.17 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 0.1 M Bis-Tris Propane (BTP), pH 6.5) 0.2 M KSCN 17.5 % polyethylene glycol 3,400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1.008 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Aug 26, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.008 Å / Relative weight: 1
ReflectionResolution: 2.65→47.612 Å / Num. obs: 58701 / % possible obs: 99.6 % / Redundancy: 6.9 % / Net I/σ(I): 17.86
Reflection shellResolution: 2.65→2.745 Å / Redundancy: 6.8 % / Mean I/σ(I) obs: 0.96 / % possible all: 98.67

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Processing

Software
NameVersionClassification
PHENIX(1.14_3211: ???)refinement
XDSdata reduction
PHASERphasing
Cootmodel building
XSCALEdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5NFV

5nfv


Resolution: 2.65→47.612 Å / SU ML: 0.47 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 36.76
RfactorNum. reflection% reflection
Rfree0.2637 2926 4.98 %
Rwork0.2482 --
obs0.249 58699 99.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.65→47.612 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10578 2266 108 30 12982
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00313403
X-RAY DIFFRACTIONf_angle_d0.61318505
X-RAY DIFFRACTIONf_dihedral_angle_d17.9047762
X-RAY DIFFRACTIONf_chiral_restr0.0392021
X-RAY DIFFRACTIONf_plane_restr0.0031969
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6501-2.69350.48241270.43162594X-RAY DIFFRACTION97
2.6935-2.740.45171420.42522651X-RAY DIFFRACTION100
2.74-2.78980.40531400.40692647X-RAY DIFFRACTION100
2.7898-2.84340.40991390.39592641X-RAY DIFFRACTION99
2.8434-2.90150.37791380.38662629X-RAY DIFFRACTION99
2.9015-2.96450.38681370.37042645X-RAY DIFFRACTION99
2.9645-3.03350.37381430.35492654X-RAY DIFFRACTION100
3.0335-3.10930.32361360.3452625X-RAY DIFFRACTION100
3.1093-3.19340.36431400.31152664X-RAY DIFFRACTION100
3.1934-3.28730.30151400.29632660X-RAY DIFFRACTION99
3.2873-3.39340.29891390.27822633X-RAY DIFFRACTION100
3.3934-3.51470.27911380.27682644X-RAY DIFFRACTION100
3.5147-3.65530.25411410.27262674X-RAY DIFFRACTION100
3.6553-3.82160.32131390.27032652X-RAY DIFFRACTION100
3.8216-4.0230.32391400.24382653X-RAY DIFFRACTION100
4.023-4.27490.25141430.23782683X-RAY DIFFRACTION100
4.2749-4.60470.20841380.21382657X-RAY DIFFRACTION100
4.6047-5.06770.21491420.21182689X-RAY DIFFRACTION100
5.0677-5.79990.22221400.2182667X-RAY DIFFRACTION100
5.7999-7.3030.21791410.21922687X-RAY DIFFRACTION100
7.303-47.61950.19751430.1742724X-RAY DIFFRACTION100
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)
11.20180.3050.28313.4261.2522.38820.0754-0.17720.35470.1348-0.25510.8522-0.489-0.4790.08830.57920.09060.10280.6488-0.05410.59498.6412-17.2435374.2693
22.6210.31650.22192.89851.17262.61970.1479-0.2450.3677-0.0130.1556-0.7476-0.45570.3299-0.31171.198-0.13760.05170.582-0.1140.9086129.14693.6047396.5583
30.95430.82860.18453.91570.4511.4878-0.19540.40890.0088-1.22640.2210.0207-0.2291-0.0636-0.0820.84350.02360.01720.8406-0.03670.416110.9488-37.1095346.1022
41.03031.3367-0.7724.67770.54661.83310.1940.1222-0.20970.69490.1987-1.7969-0.12910.6266-0.19080.63260.0944-0.37560.8958-0.19031.3705136.0317-39.9659372.5413
51.51650.09641.31680.0764-0.27822.49470.3255-0.3646-0.42220.65440.0023-1.11070.48040.1377-0.15431.18950.168-0.77360.8125-0.03631.2839135.1684-50.5435391.8734
63.7807-0.9019-0.47881.0442-0.86111.2153-0.12630.0265-0.2125-0.70840.1853-1.2699-0.51760.8944-0.10240.6118-0.17340.30221.1644-0.13441.2391133.9779-31.7858355.197
71.57840.2041-0.64011.63481.64542.10810.5234-0.4452-0.03850.8652-0.1049-0.3575-0.3066-0.4446-0.38581.2139-0.0612-0.06110.70330.03830.5849115.9958-14.6487385.319
86.35290.93384.0831.5491-1.72376.45110.5163-0.1386-1.3786-0.01140.1077-0.04540.67530.0491-0.55432.48620.4825-1.58062.3238-0.04652.7769146.8355-27.1615404.2751
90.10180.4521-0.28634.09981.33853.13450.1892-0.14940.05070.35830.0346-0.4001-0.5888-0.0588-0.18580.82180.0760.02220.8114-0.04840.5294114.5143-21.4083376.9023
103.3601-0.0670.73532.91651.53832.97080.00191.64330.2804-1.15620.11490.5834-0.8596-0.55740.5861.96850.2247-1.0921.82630.32571.49387.6332-23.0046334.3763
111.0393-0.3014-0.30712.2221-1.16010.8263-0.39811.35620.2071-0.8902-0.02560.5312-0.19620.0791-0.39641.60830.1842-0.7161.53870.05181.047492.7714-27.0299341.6638
121.4867-0.32251.3340.21930.12442.26460.2585-0.37220.26511.00250.0222-0.41220.5681-0.0058-0.24941.70420.1369-0.42061.5580.05021.0265108.2414-43.5465380.4223
137.0970.61494.10894.3407-2.21373.9223-0.1776-0.0302-0.24940.1509-0.127-0.1314-0.59161.50570.54153.03530.6824-1.3473.1126-0.02822.0563144.5617-20.2787400.855
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 329 )
2X-RAY DIFFRACTION2chain 'A' and (resid 330 through 586 )
3X-RAY DIFFRACTION3chain 'A' and (resid 587 through 906 )
4X-RAY DIFFRACTION4chain 'A' and (resid 907 through 999 )
5X-RAY DIFFRACTION5chain 'A' and (resid 1000 through 1300 )
6X-RAY DIFFRACTION6chain 'B' and (resid -18 through 3 )
7X-RAY DIFFRACTION7chain 'B' and (resid 4 through 21 )
8X-RAY DIFFRACTION8chain 'C' and (resid -27 through -23 )
9X-RAY DIFFRACTION9chain 'C' and (resid -22 through 2 )
10X-RAY DIFFRACTION10chain 'C' and (resid 3 through 10 )
11X-RAY DIFFRACTION11chain 'D' and (resid -8 through 1 )
12X-RAY DIFFRACTION12chain 'D' and (resid 2 through 19 )
13X-RAY DIFFRACTION13chain 'D' and (resid 20 through 27 )

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