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- PDB-8rcw: Crystal structure of the Mycobacterium tuberculosis regulator Vir... -

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Basic information

Entry
Database: PDB / ID: 8rcw
TitleCrystal structure of the Mycobacterium tuberculosis regulator VirS (N-terminal fragment 4-208) in complex with the lead compound SMARt751
ComponentsHTH-type transcriptional regulator VirS
KeywordsTRANSCRIPTION / ARAC FAMILY / TUBERCULOSIS / DNA BINDING PROTEIN / IN SITU PROTEOLYSIS
Function / homology
Function and homology information


cellular response to acidic pH / transcription cis-regulatory region binding / DNA-binding transcription factor activity / positive regulation of DNA-templated transcription / cytosol
Similarity search - Function
HTH-type transcriptional regulator AraC-type, N-terminal / Arabinose-binding domain of AraC transcription regulator, N-term / Helix-turn-helix domain / DNA binding HTH domain, AraC-type / Bacterial regulatory proteins, araC family DNA-binding domain profile. / helix_turn_helix, arabinose operon control protein / Homeobox-like domain superfamily
Similarity search - Domain/homology
: / HTH-type transcriptional regulator VirS
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.692 Å
AuthorsGrosse, C. / Sigoillot, M. / Megalizzi, V. / Tanina, A. / Willand, N. / Baulard, A.R. / Wintjens, R.
Funding support Belgium, France, 2items
OrganizationGrant numberCountry
Fonds National de la Recherche Scientifique (FNRS)CR40003580 Belgium
Agence Nationale de la Recherche (ANR)ANR-20-PAMR-0005 France
CitationJournal: J.Struct.Biol. / Year: 2024
Title: Crystal structure of the Mycobacterium tuberculosis VirS regulator reveals its interaction with the lead compound SMARt751.
Authors: Grosse, C. / Sigoillot, M. / Megalizzi, V. / Tanina, A. / Willand, N. / Baulard, A.R. / Wintjens, R.
History
DepositionDec 7, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: HTH-type transcriptional regulator VirS
B: HTH-type transcriptional regulator VirS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,2424
Polymers50,6362
Non-polymers6072
Water3,747208
1
A: HTH-type transcriptional regulator VirS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,6212
Polymers25,3181
Non-polymers3031
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: HTH-type transcriptional regulator VirS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,6212
Polymers25,3181
Non-polymers3031
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)115.332, 115.332, 70.987
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number169
Space group name H-MP61
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21A

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: GLY / Beg label comp-ID: GLY / End auth comp-ID: ILE / End label comp-ID: ILE / Auth asym-ID: A / Label asym-ID: A / Auth seq-ID: 4 - 208 / Label seq-ID: 3 - 207

Dom-ID
1
2

NCS ensembles : (Details: Local NCS retraints between domains: 1 2)

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Components

#1: Protein HTH-type transcriptional regulator VirS / Virulence-regulating protein VirS


Mass: 25317.932 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Proteolytic fragment: residues 4-208
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: virS, Rv3082c, MTV013.03c / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WMJ3
#2: Chemical ChemComp-YQF / 4,4,4-tris(fluoranyl)-1-[4-(4-fluorophenyl)piperidin-1-yl]butan-1-one


Mass: 303.295 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H17F4NO / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 208 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Sequence detailsThe complete sequence of the construct used for crystallization is: ...The complete sequence of the construct used for crystallization is: MGSSHHHHHHSSGLVPRGSHMDPEEASVTSTEETLTPAQEAAETEAANKAEEEAELEAETAEQMELGSLIRATNLWGYTDLMRELGADPLPFLRRFDIPPGIEHQEDAFMSLAGFVRMLEASAAELDCPDFGLRLARWQGLGILGPVAVIARNAATLFGGLEAIGRYLYVHSPALTLTVSSTTARSNVRFGYEVTEPGIPYPLQGYELSMANAARMIRLLGGPQARARVFSFRHAQLGTDAAYREALGCTVRFGRTWCGFEVDHRLAGRPIDHADPETKRIATKYLESQYLPSDATLSERVVGLARRLLPTGQCSAEAIADQLDMHPRTLQRRLAAEGLRCHDLIERERRAQAARYLAQPGLYLSQIAVLLGYSEQSALNRSCRRWFGMTPRQYRAYGGVSGR, from which MGSSHHHHHHSSGLVPRGSHMDPEEASVTSTEETLTPAQEAAETEAANKAEEEAELEAETAEQ is an N-terminal expression tag followed by the protein uniprot entry P9WMJ3 (VIRS_MYCTU). Authors state that the protein sample was mixed with protease during crystallization, which evidently cleaved off N-terminal and C-terminal residues prior to crystal formation. the precise location of the cleavage site has not been determined. Therefore, the sequence information, as well as the values of Matthews coefficient and solvent content are based on the chain length visible in electron density.

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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: 2.33 Å3/Da / Density % sol: 47.28 %
Description: 20 microliters of protein sample (225 micromolars) was mixed with 0.2 microliters of ligand SMART751 (25 millimolars) and incubated for 20 minutes on ice before addition of 2 microliters ...Description: 20 microliters of protein sample (225 micromolars) was mixed with 0.2 microliters of ligand SMART751 (25 millimolars) and incubated for 20 minutes on ice before addition of 2 microliters of subtilisin or papain protease (1 mg/ml). The Matthews coefficient calculated based on the constructed sequence is 1.53, and solvent content 19.55%. The values shown in file are calculated based on the sequence starting from the first visible N-terminal residue in electron density, therefore they may not be accurate.
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.3 M sodium acetate trihydrate, 0.1 M Tris, pH 7.5, 8% (w/v) PEG 20,000, 8% (v/v) PEG 500 MME, pH7.5, protein:SMART751 ratio 1:1, in situ proteolysis with subtilisin, temperature 293K, vapor diffusion

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.9655 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Feb 12, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9655 Å / Relative weight: 1
ReflectionResolution: 1.692→19.295 Å / Num. obs: 51863 / % possible obs: 96.5 % / Redundancy: 10.1 % / CC1/2: 0.997 / Rmerge(I) obs: 0.17 / Rpim(I) all: 0.056 / Rrim(I) all: 0.179 / Net I/σ(I): 9.6
Reflection shellResolution: 1.692→1.767 Å / Redundancy: 9.4 % / Rmerge(I) obs: 1.68 / Num. unique obs: 2593 / CC1/2: 0.56 / Rpim(I) all: 0.576 / Rrim(I) all: 1.778 / % possible all: 77.3

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Processing

Software
NameVersionClassification
REFMAC5.8.0419refinement
autoPROC1.0.5data processing
Aimless0.7.13data scaling
STARANISOdata scaling
PHASER2.8.3phasing
autoPROC1.0.5data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.692→19.295 Å / Cor.coef. Fo:Fc: 0.951 / Cor.coef. Fo:Fc free: 0.937 / WRfactor Rfree: 0.21 / WRfactor Rwork: 0.182 / SU B: 2.238 / SU ML: 0.071 / Average fsc free: 0.9669 / Average fsc work: 0.9751 / Cross valid method: THROUGHOUT / ESU R: 0.104 / ESU R Free: 0.103
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2168 2583 4.98 %
Rwork0.1855 49280 -
all0.187 --
obs-51863 86.463 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 20.15 Å2
Baniso -1Baniso -2Baniso -3
1--0.035 Å2-0.018 Å2-0 Å2
2---0.035 Å20 Å2
3---0.115 Å2
Refinement stepCycle: LAST / Resolution: 1.692→19.295 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3172 0 42 208 3422
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0113294
X-RAY DIFFRACTIONr_bond_other_d0.0010.0163122
X-RAY DIFFRACTIONr_angle_refined_deg1.6731.6584472
X-RAY DIFFRACTIONr_angle_other_deg0.5571.5667124
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3625408
X-RAY DIFFRACTIONr_dihedral_angle_2_deg7.355.68244
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.75610496
X-RAY DIFFRACTIONr_dihedral_angle_6_deg16.40210148
X-RAY DIFFRACTIONr_chiral_restr0.0860.2480
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.024044
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02856
X-RAY DIFFRACTIONr_nbd_refined0.2210.2644
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1850.22792
X-RAY DIFFRACTIONr_nbtor_refined0.1840.21678
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.080.21735
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1060.2143
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0010.21
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.0940.28
X-RAY DIFFRACTIONr_nbd_other0.2010.230
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.120.226
X-RAY DIFFRACTIONr_mcbond_it2.2821.8741638
X-RAY DIFFRACTIONr_mcbond_other2.281.8741638
X-RAY DIFFRACTIONr_mcangle_it3.3323.3572044
X-RAY DIFFRACTIONr_mcangle_other3.3323.3572045
X-RAY DIFFRACTIONr_scbond_it3.4942.2871656
X-RAY DIFFRACTIONr_scbond_other3.4962.2881654
X-RAY DIFFRACTIONr_scangle_it5.3474.0042428
X-RAY DIFFRACTIONr_scangle_other5.3464.0032429
X-RAY DIFFRACTIONr_lrange_it6.39820.673694
X-RAY DIFFRACTIONr_lrange_other6.38520.1793658
X-RAY DIFFRACTIONr_ncsr_local_group_10.0880.056427
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AX-RAY DIFFRACTIONLocal ncs0.088410.05009
12AX-RAY DIFFRACTIONLocal ncs0.088410.05009
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
1.692-1.7350.33680.2911380.29343910.930.94427.46530.284
1.735-1.7830.2591020.25520200.25542590.9650.95949.82390.248
1.783-1.8340.2851490.25424020.25641420.9470.95961.58860.246
1.834-1.890.2821270.23530680.23740510.9460.96378.86940.222
1.89-1.9510.2531800.21434640.21639100.9590.96993.19690.196
1.951-2.0190.232030.21535830.21638200.9670.96999.10990.194
2.019-2.0940.2541750.19934560.20236370.9620.97599.8350.179
2.094-2.1790.2141650.18433690.18535340.9710.9791000.166
2.179-2.2740.2111830.17331860.17533690.9720.9821000.157
2.274-2.3830.2261440.16831150.17132590.9710.9821000.151
2.383-2.510.1951590.16429370.16530960.9770.9841000.149
2.51-2.660.2171360.17627880.17829240.9730.9811000.161
2.66-2.840.1851260.17126150.17227410.9780.9821000.161
2.84-3.0620.2341250.18924590.19125840.9690.9781000.182
3.062-3.3450.2311390.19222170.19423560.960.9781000.194
3.345-3.7260.1991310.18420580.18521890.9770.9811000.189
3.726-4.2760.15660.16718330.16619000.9830.98399.94740.181
4.276-5.1740.2041080.1615370.16216450.9810.9871000.182
5.174-7.0670.197630.18812490.18913120.9810.9811000.207
7.067-19.2950.221340.1787870.188210.9790.9841000.213

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