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- EMDB-44293: Cryo-EM structure of MraY in complex with analogue 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-44293
TitleCryo-EM structure of MraY in complex with analogue 2
Map datamap
Sample
  • Complex: Complex of MraY AA dimer with nanobody bound
    • Protein or peptide: Phospho-N-acetylmuramoyl-pentapeptide-transferase
    • Protein or peptide: MraYAA nanobody
  • Ligand: (2~{S},3~{S})-3-[(2~{S},3~{R},4~{S},5~{R})-5-(aminomethyl)-3,4-bis(oxidanyl)oxolan-2-yl]oxy-2-[[3-[[[(2~{S})-6-azanyl-2-(hexadecanoylamino)hexanoyl]amino]methyl]phenyl]methylamino]-3-[(2~{S},3~{S},4~{R},5~{R})-5-[2,4-bis(oxidanylidene)pyrimidin-1-yl]-3,4-bis(oxidanyl)oxolan-2-yl]propanoic acid
  • Ligand: water
Keywordsinhibitor / antibiotics / TRANSFERASE
Function / homology
Function and homology information


phospho-N-acetylmuramoyl-pentapeptide-transferase / phospho-N-acetylmuramoyl-pentapeptide-transferase activity / UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine:undecaprenyl-phosphate transferase activity / cell wall macromolecule biosynthetic process / phosphotransferase activity, for other substituted phosphate groups / peptidoglycan biosynthetic process / cell wall organization / regulation of cell shape / cell division / metal ion binding ...phospho-N-acetylmuramoyl-pentapeptide-transferase / phospho-N-acetylmuramoyl-pentapeptide-transferase activity / UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine:undecaprenyl-phosphate transferase activity / cell wall macromolecule biosynthetic process / phosphotransferase activity, for other substituted phosphate groups / peptidoglycan biosynthetic process / cell wall organization / regulation of cell shape / cell division / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
Phospho-N-acetylmuramoyl-pentapeptide transferase / Phospho-N-acetylmuramoyl-pentapeptide transferase, conserved site / Phospho-N-acetylmuramoyl-pentapeptide-transferase signature 1 / MraY family signature 1. / MraY family signature 2. / Glycosyl transferase, family 4 / Glycosyl transferase family 4
Similarity search - Domain/homology
Phospho-N-acetylmuramoyl-pentapeptide-transferase
Similarity search - Component
Biological speciesAquifex aeolicus VF5 (bacteria) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsHao A / Lee S-Y
Funding support United States, 1 items
OrganizationGrant numberCountry
Other privateDuke Science Technology Scholar Fund United States
CitationJournal: Nat Commun / Year: 2024
Title: Development of a natural product optimization strategy for inhibitors against MraY, a promising antibacterial target.
Authors: Kazuki Yamamoto / Toyotaka Sato / Aili Hao / Kenta Asao / Rintaro Kaguchi / Shintaro Kusaka / Radhakrishnam Raju Ruddarraju / Daichi Kazamori / Kiki Seo / Satoshi Takahashi / Motohiro ...Authors: Kazuki Yamamoto / Toyotaka Sato / Aili Hao / Kenta Asao / Rintaro Kaguchi / Shintaro Kusaka / Radhakrishnam Raju Ruddarraju / Daichi Kazamori / Kiki Seo / Satoshi Takahashi / Motohiro Horiuchi / Shin-Ichi Yokota / Seok-Yong Lee / Satoshi Ichikawa /
Abstract: MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant ...MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant bacteria. Structural optimization of these natural products is required to improve their drug-like properties for therapeutic use. However, chemical modifications of these natural products are painstaking tasks due to complex synthetic processes, which is a bottleneck in advancing natural products to the clinic. Here, we develop a strategy for a comprehensive in situ evaluation of the build-up library, which enables us to streamline the preparation of the analogue library and directly assess its biological activities. We apply this approach to a series of MraY inhibitory natural products. Through construction and evaluation of the 686-compound library, we identify promising analogues that exhibit potent and broad-spectrum antibacterial activity against highly drug-resistant strains in vitro as well as in vivo in an acute thigh infection model. Structures of the MraY-analogue complexes reveal distinct interaction patterns, suggesting that these analogues represent MraY inhibitors with unique binding modes. We further demonstrate the generality of our strategy by applying it to tubulin-binding natural products to modulate their tubulin polymerization activities.
History
DepositionMar 26, 2024-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44293.png

Downloads & links

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Map

FileDownload / File: emd_44293.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.619997 - 1.5667046
Average (Standard dev.)0.0015025014 (±0.035831407)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half A

Fileemd_44293_half_map_1.map
Annotationhalf A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half B

Fileemd_44293_half_map_2.map
Annotationhalf B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of MraY AA dimer with nanobody bound

EntireName: Complex of MraY AA dimer with nanobody bound
Components
  • Complex: Complex of MraY AA dimer with nanobody bound
    • Protein or peptide: Phospho-N-acetylmuramoyl-pentapeptide-transferase
    • Protein or peptide: MraYAA nanobody
  • Ligand: (2~{S},3~{S})-3-[(2~{S},3~{R},4~{S},5~{R})-5-(aminomethyl)-3,4-bis(oxidanyl)oxolan-2-yl]oxy-2-[[3-[[[(2~{S})-6-azanyl-2-(hexadecanoylamino)hexanoyl]amino]methyl]phenyl]methylamino]-3-[(2~{S},3~{S},4~{R},5~{R})-5-[2,4-bis(oxidanylidene)pyrimidin-1-yl]-3,4-bis(oxidanyl)oxolan-2-yl]propanoic acid
  • Ligand: water

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Supramolecule #1: Complex of MraY AA dimer with nanobody bound

SupramoleculeName: Complex of MraY AA dimer with nanobody bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: Phospho-N-acetylmuramoyl-pentapeptide-transferase

MacromoleculeName: Phospho-N-acetylmuramoyl-pentapeptide-transferase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: phospho-N-acetylmuramoyl-pentapeptide-transferase
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Molecular weightTheoretical: 40.954684 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPAVPRMLYQ LALLLKDYWF AFNVLKYITF RSFTAVLIAF FLTLVLSPSF INRLRKIQRL FGGYVREYTP ESHEVKKYTP TMGGIVILI VVTLSTLLLM RWDIKYTWVV LLSFLSFGTI GFWDDYVKLK NKKGISIKTK FLLQVLSASL ISVLIYYWAD I DTILYFPF ...String:
GPAVPRMLYQ LALLLKDYWF AFNVLKYITF RSFTAVLIAF FLTLVLSPSF INRLRKIQRL FGGYVREYTP ESHEVKKYTP TMGGIVILI VVTLSTLLLM RWDIKYTWVV LLSFLSFGTI GFWDDYVKLK NKKGISIKTK FLLQVLSASL ISVLIYYWAD I DTILYFPF FKELYVDLGV LYLPFAVFVI VGSANAVNLT DGLDGLAIGP AMTTATALGV VAYAVGHSKI AQYLNIPYVP YA GELTVFC FALVGAGLGF LWFNSFPAQM FMGDVGSLSI GASLATVALL TKSEFIFAVA AGVFVFETIS VILQIIYFRW TGG KRLFKR APFHHHLELN GLPEPKIVVR MWIISILLAI IAISMLKLR

UniProtKB: Phospho-N-acetylmuramoyl-pentapeptide-transferase

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Macromolecule #2: MraYAA nanobody

MacromoleculeName: MraYAA nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 15.057651 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MVPDVQLQES GGGLVQTGGS LTLSCATSGR SFSLYAMAWF RQAPGKEREF VAGVSRRGNT AYADAVKGRF TISRDNAANT VYLQMTSLK PEDTAVYFCA AFRVAVTTYT SQQANEYNYW GQGTQVTVSS LEHHHHHH

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Macromolecule #3: (2~{S},3~{S})-3-[(2~{S},3~{R},4~{S},5~{R})-5-(aminomethyl)-3,4-bi...

MacromoleculeName: (2~{S},3~{S})-3-[(2~{S},3~{R},4~{S},5~{R})-5-(aminomethyl)-3,4-bis(oxidanyl)oxolan-2-yl]oxy-2-[[3-[[[(2~{S})-6-azanyl-2-(hexadecanoylamino)hexanoyl]amino]methyl]phenyl]methylamino]-3- ...Name: (2~{S},3~{S})-3-[(2~{S},3~{R},4~{S},5~{R})-5-(aminomethyl)-3,4-bis(oxidanyl)oxolan-2-yl]oxy-2-[[3-[[[(2~{S})-6-azanyl-2-(hexadecanoylamino)hexanoyl]amino]methyl]phenyl]methylamino]-3-[(2~{S},3~{S},4~{R},5~{R})-5-[2,4-bis(oxidanylidene)pyrimidin-1-yl]-3,4-bis(oxidanyl)oxolan-2-yl]propanoic acid
type: ligand / ID: 3 / Number of copies: 2 / Formula: A1AI1
Molecular weightTheoretical: 934.127 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration7.7 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris hydrochloride
150.0 mMNaClSodium chloride
5.0 mMDMdecyl-maltoside
2.0 mMDTTDithiothreitol

Details: 20mM Tris-HCl, 150mM NaCl, 2mM DTT, 5mM DM
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 300 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 4 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2299 / Average exposure time: 4.6 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 277701
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelPDB ID:

5ckr


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementOverall B value: 30
Output model

PDB-9b70:
Cryo-EM structure of MraY in complex with analogue 2

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