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- PDB-7mbw: Crystal structure of TnsC(1-503)A225V -

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Basic information

Entry
Database: PDB / ID: 7mbw
TitleCrystal structure of TnsC(1-503)A225V
ComponentsTransposon Tn7 transposition protein TnsC
KeywordsDNA BINDING PROTEIN / AAA+ ATPase / Transposition / DNA binding
Function / homology
Function and homology information


transposition / DNA recombination / ATP hydrolysis activity / DNA binding / ATP binding
Similarity search - Function
Tn7 transposition regulator TnsC / Tn7 transposition regulator TnsC / AAA domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Transposon Tn7 transposition protein TnsC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3.2 Å
AuthorsShen, Y. / Guarne, A.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-155941 Canada
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structural basis for DNA targeting by the Tn7 transposon.
Authors: Yao Shen / Josue Gomez-Blanco / Michael T Petassi / Joseph E Peters / Joaquin Ortega / Alba Guarné /
Abstract: Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ ...Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.
History
DepositionApr 1, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 23, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transposon Tn7 transposition protein TnsC
B: Transposon Tn7 transposition protein TnsC
C: Transposon Tn7 transposition protein TnsC
D: Transposon Tn7 transposition protein TnsC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)241,44512
Polymers239,6394
Non-polymers1,8068
Water27015
1
A: Transposon Tn7 transposition protein TnsC
B: Transposon Tn7 transposition protein TnsC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,7226
Polymers119,8192
Non-polymers9034
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6170 Å2
ΔGint-57 kcal/mol
Surface area44700 Å2
MethodPISA
2
C: Transposon Tn7 transposition protein TnsC
D: Transposon Tn7 transposition protein TnsC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,7226
Polymers119,8192
Non-polymers9034
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6390 Å2
ΔGint-56 kcal/mol
Surface area44460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)83.780, 95.770, 313.560
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein
Transposon Tn7 transposition protein TnsC / Protein E


Mass: 59909.723 Da / Num. of mol.: 4 / Mutation: S2G, A225V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: tnsC / Production host: Escherichia coli (E. coli) / References: UniProt: P05846
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.62 Å3/Da / Density % sol: 53.14 %
Crystal growTemperature: 295 K / Method: vapor diffusion
Details: 23-28% PEG 5000 MME, 0.2M Ammonium sulfate, 3% glycerol, 0.1M MES
PH range: 6.1-6.4

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: CLSI / Beamline: 08B1-1 / Wavelength: 0.97856 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Aug 8, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97856 Å / Relative weight: 1
ReflectionResolution: 3.2→95.77 Å / Num. obs: 42690 / % possible obs: 100 % / Redundancy: 14.5 % / Biso Wilson estimate: 65.17 Å2 / CC1/2: 0.99 / Net I/σ(I): 9.7
Reflection shellResolution: 3.2→3.32 Å / Redundancy: 14.7 % / Mean I/σ(I) obs: 2.3 / Num. unique obs: 4414 / CC1/2: 0.39 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.19.2_4158: ???)refinement
Aimlessdata scaling
PDB_EXTRACT3.27data extraction
iMOSFLMdata reduction
CRANK2phasing
RefinementMethod to determine structure: SAD / Resolution: 3.2→39.5 Å / SU ML: 0.43 / Cross valid method: THROUGHOUT / σ(F): 1.37 / Phase error: 24.52 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2585 2224 5.22 %
Rwork0.2164 --
obs0.2186 42582 99.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.2→39.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15031 0 112 15 15158
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00315408
X-RAY DIFFRACTIONf_angle_d0.66720860
X-RAY DIFFRACTIONf_dihedral_angle_d24.1712095
X-RAY DIFFRACTIONf_chiral_restr0.0422378
X-RAY DIFFRACTIONf_plane_restr0.0052669
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2-3.270.30241260.28222469X-RAY DIFFRACTION100
3.27-3.350.36481160.28152521X-RAY DIFFRACTION100
3.35-3.430.34711320.28052483X-RAY DIFFRACTION100
3.43-3.520.33311390.26582488X-RAY DIFFRACTION100
3.52-3.630.30561440.23462457X-RAY DIFFRACTION100
3.63-3.740.29771430.26562497X-RAY DIFFRACTION100
3.74-3.880.26321220.22432516X-RAY DIFFRACTION100
3.88-4.030.28581530.23062468X-RAY DIFFRACTION100
4.03-4.210.23571390.20252517X-RAY DIFFRACTION100
4.21-4.440.25051580.17892498X-RAY DIFFRACTION100
4.44-4.710.22561400.16942516X-RAY DIFFRACTION100
4.71-5.080.23061310.18112531X-RAY DIFFRACTION100
5.08-5.590.24591420.19532537X-RAY DIFFRACTION100
5.59-6.390.26691520.23112551X-RAY DIFFRACTION100
6.39-8.040.23281280.21532606X-RAY DIFFRACTION100
8.04-39.50.20641590.19062703X-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)
1-0.19650.0876-0.12260.59460.38371.9298-0.09370.01780.3813-0.0944-0.0638-0.1589-0.2140.24230.13920.35250.0038-0.02320.3576-0.00750.565217.2811.25145.325
24.7564-0.28411.75341.1806-0.28211.4103-0.12910.07310.2773-0.1824-0.0894-0.52530.03740.43490.17950.33460.04540.10260.54980.10370.426621.902-2.67438.788
35.102-2.23790.72825.3987-2.56895.8799-0.08720.0698-0.438-0.69120.12930.04010.47510.1891-0.06220.3161-0.08780.04070.26140.0190.2044-2.321-14.60231.746
44.1664-3.71491.51355.0186-0.6912.2376-0.24380.4380.1899-0.8685-0.03790.3826-0.2688-0.32670.20810.7443-0.0914-0.20590.66990.19580.5813-17.1533.93313.485
50.5582-1.0336-2.69511.12032.39294.3665-0.02540.1019-0.14570.1168-0.47720.9205-0.656-0.51540.48310.83710.22680.13030.8467-0.16840.87585.799-4.006-34.49
61.7746-0.8697-0.07074.40221.77861.8719-0.1397-0.23240.53210.65550.1053-0.0513-0.6116-0.30840.00550.88830.1250.02710.5270.01390.455920.7540.419-36.877
74.91611.59673.16313.68261.21747.8768-0.0107-0.2774-0.12460.5949-0.019-0.1841-0.40260.22760.03730.33710.0862-0.0160.25820.080.252832.348-23.409-45.288
87.38026.25052.30627.36342.80072.4047-0.26450.6392-0.3864-0.48210.32820.10610.021-0.1459-0.05310.27740.0164-0.10690.32450.04170.370117.008-35.135-67.641
92.86170.11261.530.93280.72392.2663-0.4475-0.42450.45530.08560.0737-0.5583-0.14450.06840.3510.312-0.0513-0.00620.3859-0.06540.560627.50912.15183.588
103.3387-1.49121.40452.378-1.30191.3265-0.1604-0.1278-0.39150.07480.11360.09610.0913-0.04270.03760.3161-0.04290.09860.2886-0.07220.243518.756-0.4181.611
113.83761.032-1.42253.6471-0.86147.5022-0.08550.1203-0.1562-0.06430.08240.67-0.0131-0.5821-0.03430.17660.0388-0.00810.2513-0.04230.4109-5.4849.67171.304
123.7667-0.27334.94592.0918-0.32477.1149-0.06630.05170.1075-0.27180.01590.6629-0.1338-0.49950.10720.34360.08540.04290.605-0.03880.6121-17.3149.81338.3
130.2938-0.55070.52040.89090.57191.28530.02420.39650.0786-0.5121-0.3805-0.376-0.0865-0.05970.41740.9891-0.07740.32840.81490.01010.68574.955.8945.324
141.9317-0.766-1.0532.41831.09362.55510.1049-0.26380.04441.06970.3077-0.6620.42120.4572-0.34831.3551-0.07970.02160.9389-0.0320.621117.702-0.3841.115
154.53050.67081.96220.8463-0.9163.48420.4860.1799-0.7215-0.0513-0.1145-0.36021.61240.6585-0.27012.11980.2630.42240.798-0.05611.01019.016-25.597-7.816
161.5367-0.5218-0.26735.79854.36896.4246-0.3208-0.4826-0.0910.20230.40470.5240.14630.2665-0.14370.36380.01010.07730.5020.15750.53438.902-36.8-43.419
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN B AND RESID 3:122 )B3 - 122
2X-RAY DIFFRACTION2( CHAIN B AND RESID 123:324 )B123 - 324
3X-RAY DIFFRACTION3( CHAIN B AND RESID 325:403 )B325 - 403
4X-RAY DIFFRACTION4( CHAIN B AND RESID 404:485 )B404 - 485
5X-RAY DIFFRACTION5( CHAIN D AND RESID 3:122 )D3 - 122
6X-RAY DIFFRACTION6( CHAIN D AND RESID 129:324 )D129 - 324
7X-RAY DIFFRACTION7( CHAIN D AND RESID 325:403 )D325 - 403
8X-RAY DIFFRACTION8( CHAIN D AND RESID 404:486 )D404 - 486
9X-RAY DIFFRACTION9( CHAIN A AND RESID 3:122 )A3 - 122
10X-RAY DIFFRACTION10( CHAIN A AND RESID 123:324 )A123 - 324
11X-RAY DIFFRACTION11( CHAIN A AND RESID 325:403 )A325 - 403
12X-RAY DIFFRACTION12( CHAIN A AND RESID 404:487 )A404 - 487
13X-RAY DIFFRACTION13( CHAIN C AND RESID 5:122 )C5 - 122
14X-RAY DIFFRACTION14( CHAIN C AND RESID 123:324 )C123 - 324
15X-RAY DIFFRACTION15( CHAIN C AND RESID 325:403 )C325 - 403
16X-RAY DIFFRACTION16( CHAIN C AND ( RESID 404:483 OR RESID 484:484 ) )C404 - 483
17X-RAY DIFFRACTION16( CHAIN C AND ( RESID 404:483 OR RESID 484:484 ) )C484

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