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- PDB-9hpn: Corynebacterium glutamicum PS2 S-layer -

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Basic information

Entry
Database: PDB / ID: 9hpn
TitleCorynebacterium glutamicum PS2 S-layer
ComponentsPS2
KeywordsSTRUCTURAL PROTEIN / S-layer / PS2 / C. glutamicum
Function / homologymembrane / PS2
Function and homology information
Biological speciesCorynebacterium glutamicum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsIsbilir, B. / Bharat, T.
Funding support United Kingdom, France, European Union, 6items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_1201/31 United Kingdom
Wellcome Trust225317/Z/22/Z United Kingdom
Human Frontier Science Program (HFSP)RGY0074/2021 France
European Molecular Biology Organization (EMBO)YIP 2021European Union
Leverhulme TrustPhilip Leverhulme Prize United Kingdom
The Lister Institute of Preventive MedicineLister Prize United Kingdom
CitationJournal: PLoS Biol / Year: 2025
Title: Mapping the ultrastructural topology of the corynebacterial cell surface.
Authors: Buse Isbilir / Anna Yeates / Vikram Alva / Tanmay A M Bharat /
Abstract: Corynebacterium glutamicum is a diderm bacterium extensively used in the industrial-scale production of amino acids. Corynebacteria belong to the bacterial family Mycobacteriaceae, which is ...Corynebacterium glutamicum is a diderm bacterium extensively used in the industrial-scale production of amino acids. Corynebacteria belong to the bacterial family Mycobacteriaceae, which is characterized by a highly unusual cell envelope with an outer membrane consisting of mycolic acids, called mycomembrane. The mycomembrane is further coated by a surface (S-)layer array in C. glutamicum, making this cell envelope highly distinctive. Despite the biotechnological significance of C. glutamicum and biomedical significance of mycomembrane-containing pathogens, ultrastructural and molecular details of its distinctive cell envelope remain poorly characterized. To address this, we investigated the cell envelope of C. glutamicum using electron cryotomography and cryomicroscopy of focused ion beam-milled single and dividing cells. Our cellular imaging allowed us to map the different components of the cell envelope onto the tomographic density. Our data reveal that C. glutamicum has a variable cell envelope, with the S-layer decorating the mycomembrane in a patchy manner. We further isolated and resolved the structure of the S-layer at 3.1 Å-resolution using single particle electron cryomicroscopy. Our structure shows that the S-layer of C. glutamicum is composed of a hexagonal array of the PS2 protein, which interacts directly with the mycomembrane via an anchoring segment containing a coiled-coil motif. Bioinformatic analyses revealed that the PS2 S-layer is sparsely yet exclusively present within the Corynebacterium genus and absent in other genera of the Mycobacteriaceae family, suggesting distinct evolutionary pathways in the development of their cell envelopes. Our structural and cellular data collectively provide a topography of the unusual C. glutamicum cell surface, features of which are shared by many pathogenic and microbiome-associated bacteria, as well as by several industrially significant bacterial species.
History
DepositionDec 13, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 29, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PS2
B: PS2
C: PS2
D: PS2
E: PS2
F: PS2


Theoretical massNumber of molelcules
Total (without water)324,2356
Polymers324,2356
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "F"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "A"
d_5ens_1chain "E"
d_6ens_1chain "D"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: THR / Beg label comp-ID: THR / End auth comp-ID: LYS / End label comp-ID: LYS / Auth seq-ID: 33 - 445 / Label seq-ID: 33 - 445

Dom-IDAuth asym-IDLabel asym-ID
d_1FF
d_2BB
d_3CC
d_4AA
d_5EE
d_6DD

NCS oper:
IDCodeMatrixVector
1given(-0.500044505907, -0.865999703146, 7.91388553627E-5), (0.865999700976, -0.500044511042, -6.99045894741E-5), (0.000100110303967, 3.35788191754E-5, 0.999999994425)520.417065493, 139.50580423, -0.0504775579236
2given(-0.500242865769, 0.865884560726, -0.00100137070869), (-0.865883303307, -0.500243852108, -0.0014810410436), (-0.00178334011416, 0.000126189961108, 0.999998401886)139.696126614, 520.843943726, 0.493668607786
3given(-0.99999579159, -0.00079793079343, -0.00278928474501), (0.000793164069314, -0.999998224026, 0.0017096300333), (-0.00279064395777, 0.00170741047804, 0.999994648514)440.72768737, 439.406603482, 0.38418598286
4given(0.49993402295, 0.866063133957, 0.000787843771917), (-0.866061038322, 0.499934565078, -0.00192575720876), (-0.00206169765692, 0.00028043075346, 0.999997835378)-80.7548422618, 300.911412781, 0.530859147726
5given(0.500142505434, -0.86594301617, 0.000408662087306), (0.86594151669, 0.500142434035, 0.00168384990189), (-0.00166250731387, -0.000488287441013, 0.999998498821)300.337921979, -80.9303905523, 0.598806648918

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Components

#1: Protein
PS2


Mass: 54039.098 Da / Num. of mol.: 6 / Source method: isolated from a natural source
Details: PS2 hexamer from Corynebacterium glutamicum 541 (ATCC-13058)
Source: (natural) Corynebacterium glutamicum (bacteria) / References: UniProt: Q6QUU5
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Corynebacterium glutamicum PS2 S-layer / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.0539 MDa / Experimental value: NO
Source (natural)Organism: Corynebacterium glutamicum ATCC 13058 (bacteria)
Buffer solutionpH: 7.5
Details: 50 mM HEPES pH=7.5, 150 mM NaCl, 1 mM MgCl2, 2 mM CaCl2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHepesC8H18N2O4S1
2150 mMSodium chlorideNaCl1
31 mMMagnesium chlorideMgCl21
42 mMCalcium chlorideCaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K
Details: 3.5 uL of resuspended sample was applied to a freshly glow discharged Quantifoil R2/2 Cu/Rh 200 mesh grid and plunge-frozen into liquid ethane maintained at -178 C, using Vitrobot Mark IV ...Details: 3.5 uL of resuspended sample was applied to a freshly glow discharged Quantifoil R2/2 Cu/Rh 200 mesh grid and plunge-frozen into liquid ethane maintained at -178 C, using Vitrobot Mark IV after a wait time of 40 seconds, with -5 blot force and 2.5 seconds blot time.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1600 nm / Calibrated defocus min: 1600 nm / Calibrated defocus max: 2200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
2PHENIX1.21.2_5419model refinement
3EPU3.7.1image acquisition
5cryoSPARC4.5.3CTF correction
11cryoSPARC4.5.3final Euler assignment
12cryoSPARC4.5.3classification
13cryoSPARC4.5.33D reconstruction
Image processingDetails: Data collected with the microscope stage tilted by 30 degrees
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50974 / Algorithm: FOURIER SPACE / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 67.76 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002119632
ELECTRON MICROSCOPYf_angle_d0.424526712
ELECTRON MICROSCOPYf_chiral_restr0.03373006
ELECTRON MICROSCOPYf_plane_restr0.00293606
ELECTRON MICROSCOPYf_dihedral_angle_d3.69472724
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2FFELECTRON MICROSCOPYNCS constraints2.26815763785E-10
ens_1d_3FFELECTRON MICROSCOPYNCS constraints1.06386707008E-12
ens_1d_4FFELECTRON MICROSCOPYNCS constraints3.55281486754E-10
ens_1d_5FFELECTRON MICROSCOPYNCS constraints1.77251695031E-12
ens_1d_6FFELECTRON MICROSCOPYNCS constraints1.11459319837E-12

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