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- EMDB-44328: Cryo-EM structure of TetR regulator Mce3R from Mycobacterium tube... -

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Basic information

Entry
Database: EMDB / ID: EMD-44328
TitleCryo-EM structure of TetR regulator Mce3R from Mycobacterium tuberculosis bound to a DNA oligonucleotide
Map data
Sample
  • Complex: TetR regulator Mce3R from Mycobacterium tuberculosis bound to a DNA oligonucleotide
    • Protein or peptide: Transcriptional repressor Mce3R
    • DNA: DNA (29-MER)
    • DNA: DNA (29-MER)
KeywordsTetR-like repressor stress resistance / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


regulation of lipid metabolic process / lipid catabolic process / transcription cis-regulatory region binding / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / DNA binding
Similarity search - Function
: / Bacterial regulatory proteins, tetR family / DNA-binding HTH domain, TetR-type / TetR-type HTH domain profile. / Homeobox-like domain superfamily
Similarity search - Domain/homology
Transcriptional repressor Mce3R
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsPanagoda N / Sampson N
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI134054 United States
CitationJournal: ACS Chem Biol / Year: 2024
Title: Mce3R TetR-like Repressor Forms an Asymmetric Four-Helix Bundle and Binds a Nonpalindrome Sequence†.
Authors: Navanjalee T Panagoda / Gábor Balázsi / Nicole S Sampson /
Abstract: (), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for 's adaptation to the human host environment. Our study focuses on one ... (), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for 's adaptation to the human host environment. Our study focuses on one notable repressor, Mce3R, composed of an unusual double TFR motif. Mce3R-regulated genes encode enzymes implicated in cholesterol metabolism, resistance against reactive oxygen species, and lipid transport activities important for survival and persistence in the host and for the cellular activity of a 6-azasteroid derivative. Here, we present the structure of Mce3R bound to its DNA operator, unveiling a unique asymmetric assembly previously unreported. We obtained a candidate DNA-binding motif through MEME motif analysis, comparing intergenic regions of orthologues and identifying nonpalindromic regions conserved between orthologues. Using an electrophoretic mobility shift assay (EMSA), we confirmed that Mce3R binds to a 123-bp sequence that includes the predicted motif. Using scrambled DNA and DNA oligonucleotides of varying lengths with sequences from the upstream region of the () operon, we elucidated the operator region to be composed of two Mce3R binding sites, each a 25-bp asymmetric sequence separated by 53 bp. Mce3R binds with a higher affinity to the downstream site with a of 2.4 ± 0.7 nM. The cryo-EM structure of Mce3R bound to the 123-bp sequence was refined to a resolution of 2.51 Å. Each Mce3R monomer comprises 21 α-helices (α1-α21) folded into an asymmetric TFR-like structure with a core asymmetric four-helix bundle. This complex has two nonidentical HTH motifs and a single ligand-binding domain. The two nonidentical HTHs from each TFR bind within the high-affinity, nonpalindromic operator motif, with Arg53 and Lys262 inserted into the major groove. Site-directed mutagenesis of Arg53 to alanine abrogated DNA binding, validating the Mce3R/DNA structure obtained. Among 811,645 particles, 63% were Mce3R homodimer bound to two duplex oligonucleotides. Mce3R homodimerizes primarily through α15, and each monomer binds to an identical site in the DNA duplex oligonucleotide.
History
DepositionMar 28, 2024-
Header (metadata) releaseNov 13, 2024-
Map releaseNov 13, 2024-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44328.png

Downloads & links

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Map

FileDownload / File: emd_44328.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 448 pix.
= 425.6 Å		0.95 Å/pix.
x 448 pix.
= 425.6 Å		0.95 Å/pix.
x 448 pix.
= 425.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.125
Minimum - Maximum-0.4250335 - 1.1502087
Average (Standard dev.)-0.000093531205 (±0.015451755)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 425.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44328_msk_1.map
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Additional map: #1

Fileemd_44328_additional_1.map
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Half map: #2

Fileemd_44328_half_map_1.map
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Half map: #1

Fileemd_44328_half_map_2.map
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Sample components

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Entire : TetR regulator Mce3R from Mycobacterium tuberculosis bound to a D...

EntireName: TetR regulator Mce3R from Mycobacterium tuberculosis bound to a DNA oligonucleotide
Components
  • Complex: TetR regulator Mce3R from Mycobacterium tuberculosis bound to a DNA oligonucleotide
    • Protein or peptide: Transcriptional repressor Mce3R
    • DNA: DNA (29-MER)
    • DNA: DNA (29-MER)

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Supramolecule #1: TetR regulator Mce3R from Mycobacterium tuberculosis bound to a D...

SupramoleculeName: TetR regulator Mce3R from Mycobacterium tuberculosis bound to a DNA oligonucleotide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Protein was prepared from a maltose-binding protein fusion construct and proteolytic cleavage with Tev protease.
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)

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Macromolecule #1: Transcriptional repressor Mce3R

MacromoleculeName: Transcriptional repressor Mce3R / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 44.400602 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MASVAQPVRR RPKDRKKQIL DQAVGLFIER GFHSVKLEDI AEAAGVTARA LYRHYDNKQA LLAEAIRTGQ DQYQSARRLT EGETEPTPR PLNADLEDLI AAAVASRALT VLWQREARYL NEDDRTAVRR RINAIVAGMR DSVLLEVPDL SPQHSELRAW A VSSTLTSL ...String:
MASVAQPVRR RPKDRKKQIL DQAVGLFIER GFHSVKLEDI AEAAGVTARA LYRHYDNKQA LLAEAIRTGQ DQYQSARRLT EGETEPTPR PLNADLEDLI AAAVASRALT VLWQREARYL NEDDRTAVRR RINAIVAGMR DSVLLEVPDL SPQHSELRAW A VSSTLTSL GRHSLSLPGE ELKKLLYQAC MAAARTPPVC ELPPLPAGDA ARDEADVLFS RYETLLAAGA RLFRAQGYPA VN TSEIGKG AGIAGPGLYR SFSSKQAILD ALIRRLDEWR CLECIRALRA NQQAAQRLRG LVQGHVRISL DAPDLVAVSV TEL SHASVE VRDGYLRNQG DREAVWIDLI GKLVPATSVA QGRLLVAAAI SFIEDVARTW HLTRYAGVAD EISGLALAIL TSGA GNLLR A

UniProtKB: Transcriptional repressor Mce3R

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Macromolecule #2: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 37.845172 KDa
SequenceString: (DG)(DC)(DC)(DC)(DC)(DG)(DC)(DG)(DC)(DT) (DA)(DT)(DA)(DG)(DG)(DA)(DT)(DA)(DC)(DT) (DA)(DG)(DC)(DA)(DA)(DG)(DA)(DT)(DA) (DC)(DA)(DT)(DC)(DA)(DT)(DA)(DG)(DC)(DC) (DA) (DA)(DT)(DA)(DT)(DA)(DT) ...String:
(DG)(DC)(DC)(DC)(DC)(DG)(DC)(DG)(DC)(DT) (DA)(DT)(DA)(DG)(DG)(DA)(DT)(DA)(DC)(DT) (DA)(DG)(DC)(DA)(DA)(DG)(DA)(DT)(DA) (DC)(DA)(DT)(DC)(DA)(DT)(DA)(DG)(DC)(DC) (DA) (DA)(DT)(DA)(DT)(DA)(DT)(DG)(DC) (DC)(DA)(DG)(DT)(DT)(DT)(DG)(DC)(DA)(DT) (DT)(DG) (DC)(DT)(DA)(DT)(DT)(DT)(DA) (DC)(DC)(DG)(DA)(DT)(DC)(DA)(DG)(DT)(DT) (DG)(DT)(DC) (DC)(DA)(DA)(DG)(DC)(DA) (DA)(DT)(DC)(DG)(DC)(DG)(DT)(DA)(DT)(DT) (DG)(DG)(DC)(DT) (DA)(DT)(DG)(DG)(DA) (DC)(DA)(DT)(DC)(DA)(DG)(DC)(DG)(DG)(DT) (DT)(DC)(DT)(DG)(DC) (DC)(DG)(DC)

GENBANK: GENBANK: AL123456.3

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Macromolecule #3: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 38.063328 KDa
SequenceString: (DG)(DC)(DG)(DG)(DC)(DA)(DG)(DA)(DA)(DC) (DC)(DG)(DC)(DT)(DG)(DA)(DT)(DG)(DT)(DC) (DC)(DA)(DT)(DA)(DG)(DC)(DC)(DA)(DA) (DT)(DA)(DC)(DG)(DC)(DG)(DA)(DT)(DT)(DG) (DC) (DT)(DT)(DG)(DG)(DA)(DC) ...String:
(DG)(DC)(DG)(DG)(DC)(DA)(DG)(DA)(DA)(DC) (DC)(DG)(DC)(DT)(DG)(DA)(DT)(DG)(DT)(DC) (DC)(DA)(DT)(DA)(DG)(DC)(DC)(DA)(DA) (DT)(DA)(DC)(DG)(DC)(DG)(DA)(DT)(DT)(DG) (DC) (DT)(DT)(DG)(DG)(DA)(DC)(DA)(DA) (DC)(DT)(DG)(DA)(DT)(DC)(DG)(DG)(DT)(DA) (DA)(DA) (DT)(DA)(DG)(DC)(DA)(DA)(DT) (DG)(DC)(DA)(DA)(DA)(DC)(DT)(DG)(DG)(DC) (DA)(DT)(DA) (DT)(DA)(DT)(DT)(DG)(DG) (DC)(DT)(DA)(DT)(DG)(DA)(DT)(DG)(DT)(DA) (DT)(DC)(DT)(DT) (DG)(DC)(DT)(DA)(DG) (DT)(DA)(DT)(DC)(DC)(DT)(DA)(DT)(DA)(DG) (DC)(DG)(DC)(DG)(DG) (DG)(DG)(DC)

GENBANK: GENBANK: AL123456.3

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
VitrificationCryogen name: NITROGEN

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 135814
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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