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- PDB-8h9d: Crystal structure of Cas12a protein -

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Basic information

Entry
Database: PDB / ID: 8h9d
TitleCrystal structure of Cas12a protein
Components
  • (Cas12A) x 2
  • RNA (5'-R(P*AP*AP*UP*UP*UP*CP*UP*AP*CP*UP*AP*AP*GP*UP*GP*UP*AP*GP*AP*UP*C)-3')
KeywordsDNA BINDING PROTEIN / CRISPR-Cas / Cas12a
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesLachnospiraceae bacterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 Å
AuthorsJianwei, L. / Jobichen, C. / Sivaraman, J.
Funding support Singapore, 2items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)R-154-000-C07-114 Singapore
Ministry of Education (MoE, Singapore)R154-000-A39-112 Singapore
CitationJournal: PLoS Biol / Year: 2023
Title: Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.
Authors: Li Jianwei / Chacko Jobichen / Satoru Machida / Sun Meng / Randy J Read / Chen Hongying / Shi Jian / Yuren Adam Yuan / J Sivaraman /
Abstract: Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium ...Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.
History
DepositionOct 25, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 29, 2023Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Cas12A
A: Cas12A
G: RNA (5'-R(P*AP*AP*UP*UP*UP*CP*UP*AP*CP*UP*AP*AP*GP*UP*GP*UP*AP*GP*AP*UP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)289,3196
Polymers289,1743
Non-polymers1453
Water1086
1
B: Cas12A


Theoretical massNumber of molelcules
Total (without water)141,2011
Polymers141,2011
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area50390 Å2
MethodPISA
2
A: Cas12A
G: RNA (5'-R(P*AP*AP*UP*UP*UP*CP*UP*AP*CP*UP*AP*AP*GP*UP*GP*UP*AP*GP*AP*UP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,1185
Polymers147,9742
Non-polymers1453
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4990 Å2
ΔGint-33 kcal/mol
Surface area55670 Å2
MethodPISA
Unit cell
Length a, b, c (Å)235.701, 85.628, 139.303
Angle α, β, γ (deg.)90.00, 110.15, 90.00
Int Tables number5
Space group name H-MC121

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Components

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Protein , 2 types, 2 molecules BA

#1: Protein Cas12A


Mass: 141200.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium (bacteria)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: Protein Cas12A


Mass: 141313.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium (bacteria)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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RNA chain , 1 types, 1 molecules G

#3: RNA chain RNA (5'-R(P*AP*AP*UP*UP*UP*CP*UP*AP*CP*UP*AP*AP*GP*UP*GP*UP*AP*GP*AP*UP*C)-3')


Mass: 6659.973 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Lachnospiraceae bacterium (bacteria)

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Non-polymers , 3 types, 9 molecules

#4: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#5: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 18.015 Da / Num. of mol.: 6 / 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: 2.67 Å3/Da / Density % sol: 53.91 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop
Details: 10 mM MgCl2, 0.1 M Sodium cacodylate pH 6.8, 17% PEG1000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSRRC / Beamline: TPS 05A / Wavelength: 0.987 Å
DetectorType: RAYONIX MX300-HS / Detector: CCD / Date: Apr 25, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 3→50 Å / Num. obs: 51432 / % possible obs: 99.5 % / Redundancy: 6.6 % / Rsym value: 0.16 / Net I/σ(I): 15.6
Reflection shellResolution: 3→3.1 Å / Num. unique obs: 3567 / Rsym value: 0.93

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Processing

Software
NameVersionClassification
PHENIX(1.19.2_4158: ???)refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5ID6

5id6


Resolution: 3.1→29.98 Å / SU ML: 0.42 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 27.73 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2524 1846 3.89 %
Rwork0.2321 --
obs0.2329 47506 99.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.1→29.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16530 443 7 6 16986
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00417335
X-RAY DIFFRACTIONf_angle_d0.79523437
X-RAY DIFFRACTIONf_dihedral_angle_d9.8022471
X-RAY DIFFRACTIONf_chiral_restr0.0452633
X-RAY DIFFRACTIONf_plane_restr0.0072925
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.180.33181380.31293429X-RAY DIFFRACTION99
3.18-3.280.32331420.31343502X-RAY DIFFRACTION100
3.28-3.380.3071410.30873485X-RAY DIFFRACTION100
3.38-3.50.28751420.29033486X-RAY DIFFRACTION100
3.5-3.640.28081420.27533504X-RAY DIFFRACTION100
3.64-3.810.26361410.2683498X-RAY DIFFRACTION100
3.81-4.010.28071420.2523500X-RAY DIFFRACTION100
4.01-4.260.28561410.23453517X-RAY DIFFRACTION100
4.26-4.590.24861430.22163502X-RAY DIFFRACTION100
4.59-5.050.2221420.21393522X-RAY DIFFRACTION100
5.05-5.770.25741440.22943543X-RAY DIFFRACTION100
5.77-7.260.26651430.23913549X-RAY DIFFRACTION100
7.26-29.980.20471450.18053623X-RAY DIFFRACTION100

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