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- PDB-8ae1: Structure of trimeric SlpA outer membrane protein -

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Basic information

Entry
Database: PDB / ID: 8ae1
TitleStructure of trimeric SlpA outer membrane protein
ComponentsS-layer protein SlpA
KeywordsSTRUCTURAL PROTEIN / SlpA protein
Function / homology
Function and homology information


porin activity / pore complex / monoatomic ion transport / cell outer membrane / lipid binding
Similarity search - Function
: / S-layer protein SlpA, beta-barrel / S-layer homology domain / S-layer homology domain / S-layer homology (SLH) domain profile.
Similarity search - Domain/homology
Outer membrane protein SlpA
Similarity search - Component
Biological speciesDeinococcus radiodurans (radioresistant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
Authorsvon Kuegelgen, A. / Bharat, T.A.M.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Other privateVallee Scholarship
Leverhulme TrustPhilip Leverhulme Prize United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: A multidomain connector links the outer membrane and cell wall in phylogenetically deep-branching bacteria.
Authors: Andriko von Kügelgen / Sofie van Dorst / Vikram Alva / Tanmay A M Bharat /
Abstract: is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme ... is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in and related bacteria. Here, we report a electron cryomicroscopy (cryo-EM) structure of the abundant OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded β-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Furthermore, using protein structure prediction and sequence-based bioinformatic analysis, we show that SlpA-like putative OM-PG connector proteins are widespread in phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures with fluorescence and electron microscopy of cell envelopes of wild-type and mutant bacterial strains, we report a model for the cell surface of . Our results will have important implications for understanding the cell surface organization and hyperstability of and related bacteria and the evolutionary transition between Gram-negative and Gram-positive bacteria.
History
DepositionJul 12, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: S-layer protein SlpA
B: S-layer protein SlpA
C: S-layer protein SlpA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)372,22821
Polymers371,5063
Non-polymers72118
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein S-layer protein SlpA


Mass: 123835.367 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Deinococcus radiodurans (radioresistant)
Strain: ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422
References: UniProt: Q9RRB6
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Structure of trimeric SlpA protein / Type: COMPLEX / Details: Structure of trimeric SlpA protein / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Deinococcus radiodurans (radioresistant) / Strain: BAA-816
Buffer solutionpH: 7.5
Details: Buffer solutions were prepared fresh from sterile filtered concentrated stocksolutions. Solutions were filtered through a 0.22 um filter to avoid microbial contamination and degassed using a ...Details: Buffer solutions were prepared fresh from sterile filtered concentrated stocksolutions. Solutions were filtered through a 0.22 um filter to avoid microbial contamination and degassed using a vacuum fold pump. The pH of the HEPES stock solution was adjusted with sodium hydroxide at 4 deg C.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaCl1
30.02 % w/vDDMC24H46O111
SpecimenConc.: 4.45 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified SlpA protein after size-exclusion chromatography
Specimen supportDetails: 20 seconds, 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K
Details: Vitrobot options: Blot time 4.5 seconds, Blot force -10,1, Wait time 10 seconds, Drain time 0.5 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: EPU software with faster acquisition mode AFIS (Aberration Free Image Shift).
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: 4000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 4.8 sec. / Electron dose: 47.909 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2294
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Topaz0.2.5particle selectionResNet8 trained model
2EPUimage acquisition
4CTFFIND4.1.13CTF correctionCTFFIND4 was used as implemented in RELION 3.1
7Coot0.9.2-premodel fitting
9PHENIX1.19-4092model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
Image processingDetails: Movies were clustered into optics groups based on the XML meta-data of the data-collection software EPU (ThermoFisher) using a k-means algorithm implemented in EPU_group_AFIS (https://github. ...Details: Movies were clustered into optics groups based on the XML meta-data of the data-collection software EPU (ThermoFisher) 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 (Zheng et al., 2017) implemented in RELION3.1 (Zivanov et al., 2018). Contrast transfer functions (CTFs) of the resulting motion-corrected micrographs were estimated using CTFFIND4 (Rohou and Grigorieff, 2015).
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: 223878
Details: Initially, micrographs were denoised using TOPAZ (73) using the UNET neural network and 2893 particles were manually picked. Particle coordinates were used to train TOPAZ picker (74) in 5 ...Details: Initially, micrographs were denoised using TOPAZ (73) using the UNET neural network and 2893 particles were manually picked. Particle coordinates were used to train TOPAZ picker (74) in 5 times downsampled micrographs with the neural network architecture ResNet8 and picked particles were extracted in 4 times downsampled 128 x 128 boxes and classified using reference-free 2D classification inside RELION3.1.
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122412 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 51.67 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Best Fit
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221912
ELECTRON MICROSCOPYf_angle_d0.45329781
ELECTRON MICROSCOPYf_dihedral_angle_d3.9183207
ELECTRON MICROSCOPYf_chiral_restr0.0433222
ELECTRON MICROSCOPYf_plane_restr0.0034044

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