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- PDB-8em1: Type IIS Restriction Endonuclease PaqCI, DNA Unbound -

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Basic information

Entry
Database: PDB / ID: 8em1
TitleType IIS Restriction Endonuclease PaqCI, DNA Unbound
ComponentsPaqCI, DNA Unbound
KeywordsDNA BINDING PROTEIN / homotetramer / restriction endonuclease / DNA binding / DNA cleavage
Function / homologyUncharacterized protein
Function and homology information
Biological speciesPaucibacter aquatile (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.5 Å
AuthorsKennedy, M.A. / Stoddard, B.L.
Funding support United States, 3items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DGE-1762114 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 GM105691 United States
National Institutes of Health/Office of the Director1S10OD028581-01 United States
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.
Authors: Madison A Kennedy / Christopher J Hosford / Caleigh M Azumaya / Yvette A Luyten / Minyong Chen / Richard D Morgan / Barry L Stoddard /
Abstract: Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in ...Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.
History
DepositionSep 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 19, 2023Group: Database references / Refinement description / Category: citation / citation_author / struct_ncs_dom_lim
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id
Revision 1.2Jun 7, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Apr 3, 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: PaqCI, DNA Unbound
B: PaqCI, DNA Unbound
hetero molecules


Theoretical massNumber of molelcules
Total (without water)112,7834
Polymers112,6592
Non-polymers1242
Water1,24369
1
A: PaqCI, DNA Unbound
B: PaqCI, DNA Unbound
hetero molecules

A: PaqCI, DNA Unbound
B: PaqCI, DNA Unbound
hetero molecules


Theoretical massNumber of molelcules
Total (without water)225,5668
Polymers225,3174
Non-polymers2484
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_554-y,-x,-z-1/21
Buried area11300 Å2
ΔGint-62 kcal/mol
Surface area76420 Å2
MethodPISA
2


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2590 Å2
ΔGint-17 kcal/mol
Surface area41270 Å2
MethodPISA
Unit cell
Length a, b, c (Å)136.620, 136.620, 106.418
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and ((resid 3 through 4 and (name N...
21(chain B and (resid 3 through 28 or (resid 29...

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11TYRTYRASPASP(chain A and ((resid 3 through 4 and (name N...AA3 - 43 - 4
12PROPROLEULEU(chain A and ((resid 3 through 4 and (name N...AA2 - 5102 - 510
13PROPROLEULEU(chain A and ((resid 3 through 4 and (name N...AA2 - 5102 - 510
14PROPROLEULEU(chain A and ((resid 3 through 4 and (name N...AA2 - 5102 - 510
15PROPROLEULEU(chain A and ((resid 3 through 4 and (name N...AA2 - 5102 - 510
21TYRTYRTYRTYR(chain B and (resid 3 through 28 or (resid 29...BB3 - 283 - 28
22ASPASPSERSER(chain B and (resid 3 through 28 or (resid 29...BB29 - 3229 - 32
23TYRTYRLEULEU(chain B and (resid 3 through 28 or (resid 29...BB3 - 5103 - 510
24TYRTYRLEULEU(chain B and (resid 3 through 28 or (resid 29...BB3 - 5103 - 510
25TYRTYRLEULEU(chain B and (resid 3 through 28 or (resid 29...BB3 - 5103 - 510
26TYRTYRLEULEU(chain B and (resid 3 through 28 or (resid 29...BB3 - 5103 - 510

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Components

#1: Protein PaqCI, DNA Unbound


Mass: 56329.355 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paucibacter aquatile (bacteria) / Gene: C1O66_13805 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2N8KYF9
#2: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 69 / Source method: isolated from a natural source / Formula: H2O
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: 2.2 Å3/Da / Density % sol: 44.19 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 25% w/v polyethylene glycol 3350, 0.2 M magnesium chloride hexahydrate, and 0.1 M BIS-TRIS pH 5.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 0.9762 Å
DetectorType: DECTRIS PILATUS3 2M / Detector: PIXEL / Date: Nov 19, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9762 Å / Relative weight: 1
ReflectionResolution: 2.2→50 Å / Num. obs: 51645 / % possible obs: 100 % / Redundancy: 25.4 % / Biso Wilson estimate: 46.8 Å2 / Rmerge(I) obs: 0.1 / Rpim(I) all: 0.02 / Rrim(I) all: 0.102 / Χ2: 0.662 / Net I/σ(I): 4.6 / Num. measured all: 1313372
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.2-2.24210.9725160.880.2120.9940.42799.6
2.24-2.2822.50.85525640.9280.1820.8750.435100
2.28-2.3223.70.78925200.9440.1650.8060.435100
2.32-2.3724.10.7325460.9530.1510.7460.435100
2.37-2.4226.50.6425320.9680.1260.6520.444100
2.42-2.4826.20.55825630.9750.1110.5690.461100
2.48-2.5425.40.47525420.9810.0960.4850.472100
2.54-2.6127.20.41825520.9840.0810.4260.477100
2.61-2.6926.90.34625490.990.0680.3520.498100
2.69-2.7726.40.28925410.9930.0570.2950.524100
2.77-2.8725.20.23825800.9950.0480.2430.556100
2.87-2.9926.60.19725740.9960.0390.2010.607100
2.99-3.1225.60.15625630.9970.0310.1590.675100
3.12-3.2927.30.13125680.9980.0260.1340.754100
3.29-3.4926.80.10425880.9980.020.1060.84100
3.49-3.7625.30.08426130.9990.0170.0860.979100
3.76-4.1425.70.07126120.9990.0140.0721.012100
4.14-4.7426.80.05926190.9990.0120.0611.032100
4.74-5.9725.20.05426790.9990.0110.0550.86100
5.97-5024.30.05528240.9990.0120.0561.157100

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.5 Å49.58 Å
Translation2.5 Å49.58 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASER2.8.3phasing
PHENIX1.2refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: AlpaFold Model

Resolution: 2.5→49.58 Å / SU ML: 0.35 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 28.26 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2665 1987 5.66 %
Rwork0.2106 33137 -
obs0.2137 35124 99.4 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 126.86 Å2 / Biso mean: 55.3871 Å2 / Biso min: 30.21 Å2
Refinement stepCycle: final / Resolution: 2.5→49.58 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7276 0 8 69 7353
Biso mean--57.67 47.22 -
Num. residues----973
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2867X-RAY DIFFRACTION10.124TORSIONAL
12B2867X-RAY DIFFRACTION10.124TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.5-2.560.3071340.23292297243198
2.56-2.630.29771410.22192318245999
2.63-2.710.28681390.22562308244799
2.71-2.80.31071410.23132340248199
2.8-2.90.38121370.2552306244399
2.9-3.010.32641410.255123412482100
3.01-3.150.29441420.239923642506100
3.15-3.320.3131390.23272342248199
3.32-3.530.28291410.238123532494100
3.53-3.80.25961440.204723862530100
3.8-4.180.25071420.18623732515100
4.18-4.780.22781440.172424122556100
4.78-6.030.25741480.204524382586100
6.03-49.580.22161540.2012559271399
Refinement TLS params.Method: refined / Origin x: 34.2159 Å / Origin y: -34.09 Å / Origin z: -11.6075 Å
111213212223313233
T0.4396 Å2-0.0433 Å20.0197 Å2-0.346 Å2-0.0051 Å2--0.342 Å2
L0.3227 °20.1494 °2-0.0672 °2-0.3005 °2-0.058 °2--0.2202 °2
S0.1372 Å °-0.1423 Å °0.022 Å °0.099 Å °-0.108 Å °0.0538 Å °-0.031 Å °0.0264 Å °-0.0181 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA2 - 510
2X-RAY DIFFRACTION1allB3 - 510
3X-RAY DIFFRACTION1allS1 - 96
4X-RAY DIFFRACTION1allE1 - 2

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