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- EMDB-41304: Periplasmic map -

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Basic information

Entry
Database: EMDB / ID: EMD-41304
TitlePeriplasmic map
Map dataPeriplasmic map
Sample
  • Complex: RodA-PBP2
KeywordsPeptidoglycan / glycosyltransferase / enzyme / MEMBRANE PROTEIN
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsNygaard R / Mancia F
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM132120 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex.
Authors: Rie Nygaard / Chris L B Graham / Meagan Belcher Dufrisne / Jonathan D Colburn / Joseph Pepe / Molly A Hydorn / Silvia Corradi / Chelsea M Brown / Khuram U Ashraf / Owen N Vickery / Nicholas ...Authors: Rie Nygaard / Chris L B Graham / Meagan Belcher Dufrisne / Jonathan D Colburn / Joseph Pepe / Molly A Hydorn / Silvia Corradi / Chelsea M Brown / Khuram U Ashraf / Owen N Vickery / Nicholas S Briggs / John J Deering / Brian Kloss / Bruno Botta / Oliver B Clarke / Linda Columbus / Jonathan Dworkin / Phillip J Stansfeld / David I Roper / Filippo Mancia /
Abstract: Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular ...Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.
History
DepositionJul 21, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41304.png

Downloads & links

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Map

FileDownload / File: emd_41304.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPeriplasmic map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 332. Å		0.83 Å/pix.
x 400 pix.
= 332. Å		0.83 Å/pix.
x 400 pix.
= 332. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.29018566 - 0.54822797
Average (Standard dev.)-0.00016388434 (±0.006868241)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 332.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_41304_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_41304_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RodA-PBP2

EntireName: RodA-PBP2
Components
  • Complex: RodA-PBP2

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Supramolecule #1: RodA-PBP2

SupramoleculeName: RodA-PBP2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: Nanodisc were formed using MSP1E3D1 and POPG lipid
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 111.803 KDa

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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

Concentration0.66 mg/mL
BufferpH: 7
Component:
ConcentrationNameFormula
20.0 mMHEPES
150.0 mMSodium ChlorideNaCl
1.0 mMTCEPtris(2-carboxyethyl)phosphine
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 11120 / Average exposure time: 2.5 sec. / Average electron dose: 58.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm

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Image processing

Particle selectionNumber selected: 4415933
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.12 and 3.2) / Software - details: Local refinement / Number images used: 104553
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

4f0a


Chain - Source name: PDB / Chain - Initial model type: experimental model

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