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- PDB-8cka: Deinococcus radidurans HPI S-layer -

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Basic information

Entry
Database: PDB / ID: 8cka
TitleDeinococcus radidurans HPI S-layer
ComponentsHexagonally packed intermediate-layer surface protein
KeywordsSTRUCTURAL PROTEIN / HPI S-layer
Function / homologyS-layer / cell wall organization / Prokaryotic membrane lipoprotein lipid attachment site profile. / extracellular region / Hexagonally packed intermediate-layer surface protein
Function and homology information
Biological speciesDeinococcus radiodurans (radioresistant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.52 Å
Authorsvon Kuegelgen, A. / Yamashita, K. / Murshudov, G. / Bharat, T.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/31 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium .
Authors: Andriko von Kügelgen / Sofie van Dorst / Keitaro Yamashita / Danielle L Sexton / Elitza I Tocheva / Garib Murshudov / Vikram Alva / Tanmay A M Bharat /
Abstract: is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, due in part to its complex cell envelope encapsulated within a hyperstable surface layer (S- ... is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, due in part to its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research on this cell envelope, atomic structural details of the S-layer have remained obscure. In this study, we report the electron cryomicroscopy structure of the S-layer, showing how it is formed by the Hexagonally Packed Intermediate-layer (HPI) protein arranged in a planar hexagonal lattice. The HPI protein forms an array of immunoglobulin-like folds within the S-layer, with each monomer extending into the adjacent hexamer, resulting in a highly interconnected, stable, sheet-like arrangement. Using electron cryotomography and subtomogram averaging from focused ion beam-milled cells, we have obtained a structure of the cellular S-layer, showing how this HPI S-layer coats native membranes on the surface of cells. Our S-layer structure from the diderm bacterium shows similarities to immunoglobulin-like domain-containing S-layers from monoderm bacteria and archaea, highlighting common features in cell surface organization across different domains of life, with connotations on the evolution of immunoglobulin-based molecular recognition systems in eukaryotes.
History
DepositionFeb 14, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 23, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature / refine
Item: _em_admin.last_update / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hexagonally packed intermediate-layer surface protein
B: Hexagonally packed intermediate-layer surface protein


Theoretical massNumber of molelcules
Total (without water)198,8482
Polymers198,8482
Non-polymers00
Water00
1
A: Hexagonally packed intermediate-layer surface protein
B: Hexagonally packed intermediate-layer surface protein
x 6


Theoretical massNumber of molelcules
Total (without water)1,193,08812
Polymers1,193,08812
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
point symmetry operation5
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(0.5, -0.866025), (0.866025, 0.5), (1)298.33994, -79.93995
3generate(-0.5, -0.866025), (0.866025, -0.5), (1)516.73993, 138.46005
4generate(-1), (-1), (1)436.79999, 436.79999
5generate(-0.5, 0.866025), (-0.866025, -0.5), (1)138.46005, 516.73993
6generate(0.5, 0.866025), (-0.866025, 0.5), (1)-79.93995, 298.33994

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Components

#1: Protein Hexagonally packed intermediate-layer surface protein


Mass: 99424.000 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422) (radioresistant)
References: UniProt: P56867
Has protein modificationY

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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: Structure of Hexagonally Packed Intermediate-layer (HPI) protein
Type: ORGANELLE OR CELLULAR COMPONENT
Details: Structure of Hexagonally Packed Intermediate-layer (HPI) protein
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Deinococcus radiodurans (radioresistant) / Cellular location: extracellular
Buffer solutionpH: 7.5
Details: 50 mM HEPES/NaOH pH=7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM CaCl2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMNaClNaCl1
35 mMMgCl2MgCl21
41 mMCaCl2CaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: In vitro isolate Hexagonally Packed Intermediate-layer (HPI) layer
Specimen supportDetails: 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 283.15 K
Details: absorption for 60 sec and blotted for 5 sec with blot force -10

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 81000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 2000 nm / Calibrated defocus max: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 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 recordingAverage exposure time: 3.43 sec. / Electron dose: 53.245 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1002
EM imaging opticsEnergyfilter name: GIF Quantum LS / Chromatic aberration corrector: not used / Energyfilter slit width: 20 eV / Spherical aberration corrector: not used
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: REFMAC / Version: 5.8.0403 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Topaz0.2.5particle selectionResNet8 trained model
2RELION3.1.2particle selection
3EPUimage acquisition
5CTFFIND4.1.13CTF correctionCTFFIND4 was used as implemented in RELION 3.1
8Coot0.9.2-premodel fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14REFMAC5.8.0403model refinementServalcat v 0.3.0
Image processingDetails: Movies collected at the scope were clustered into optics groups based on the XML meta-data of the data-collection software EPU (Thermo Fisher Scientific) using a k-means algorithm ...Details: Movies collected at the scope were clustered into optics groups based on the XML meta-data of the data-collection software EPU (Thermo Fisher Scientific) using a k-means algorithm implemented in EPU_group_AFIS (https://github.com/DustinMorado/EPU_group_AFIS). Imported movies were motion-corrected, dose-weighted, and Fourier cropped (2x) with MotionCor2 implemented in RELION-3.1. Contrast transfer functions (CTFs) of the resulting motion-corrected micrographs were estimated using CTFFIND4.
CTF correctionDetails: RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 882866
Details: Initially, side views of S-layer sheets were first manually picked along the edge of the lattice using the helical picking tab in RELION while setting the helical rise to 40 Angstrom. Top ...Details: Initially, side views of S-layer sheets were first manually picked along the edge of the lattice using the helical picking tab in RELION while setting the helical rise to 40 Angstrom. Top and tilted views were manually picked at the central hexameric axis. Manually picked particles were extracted in 4x downsampled 100 x 100 boxes and classified using reference-free 2D classification inside RELION3.1. Class averages centered at a hexameric axis were used to automatically pick particles inside RELION3.1. Automatically picked particles were extracted in 4x downsampled 100x100 pixel2 boxes and classified using reference-free 2D classification. Particle coordinates belonging to class averages centered at the hexameric axis were used to train TOPAZ in 5x downsampled micrographs with the neural network architecture conv127. For the final reconstruction, particles were picked using TOPAZ and the previously trained neural network above. Additionally, top, bottom, and side views were picked using the reference-based autopicker inside RELION3.1, which TOPAZ did not readily identify. Particles were extracted in 4x downsampled 100x100 pixel2 boxes and classified using reference-free 2D classification inside RELION3.1. Particles belonging to class averages centered at the hexameric axis were combined, and particles within 30 Angstrom were removed to prevent duplication after alignment. All resulting particles were then re-extracted in 4x downsampled 100x100 pixel2 boxes.
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55345 / Algorithm: FOURIER SPACE
Details: Per-particle defocus, anisotropy magnification, and higher-order aberrations were refined inside RELION3.1, followed by another round of focused 3D auto refinement and Bayesian particle ...Details: Per-particle defocus, anisotropy magnification, and higher-order aberrations were refined inside RELION3.1, followed by another round of focused 3D auto refinement and Bayesian particle polishing. The final map was obtained from 55,345 particles and post-processed using a soft mask focused on the central hexamer, including the dimeric bridge, yielding a global resolution of 2.52 Angstrom according to the gold standard Fourier shell correlation criterion of 0.143.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 19.48 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Best Fit
RefinementResolution: 2.52→2.52 Å / Cor.coef. Fo:Fc: 0.924 / SU B: 7.928 / SU ML: 0.163 / ESU R: 0.07
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.36684 --
obs0.36684 1247499 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 75.352 Å2
Refinement stepCycle: 1 / Total: 6795
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0040.0116912
ELECTRON MICROSCOPYr_bond_other_d00.0166367
ELECTRON MICROSCOPYr_angle_refined_deg0.961.6449439
ELECTRON MICROSCOPYr_angle_other_deg0.331.56214579
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.1195913
ELECTRON MICROSCOPYr_dihedral_angle_2_deg12.499544
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.319101014
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0430.21101
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.028497
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021655
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it4.587.6563661
ELECTRON MICROSCOPYr_mcbond_other4.5767.6553661
ELECTRON MICROSCOPYr_mcangle_it7.51513.7554571
ELECTRON MICROSCOPYr_mcangle_other7.52513.7554572
ELECTRON MICROSCOPYr_scbond_it4.5987.493251
ELECTRON MICROSCOPYr_scbond_other4.5977.493252
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other7.82713.7614869
ELECTRON MICROSCOPYr_long_range_B_refined14.72288.3827693
ELECTRON MICROSCOPYr_long_range_B_other14.72288.3727694
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.54→2.606 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.563 92459 -
obs--100 %

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