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- EMDB-15378: Structure of trimeric SlpA outer membrane protein -

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Basic information

Entry
Database: EMDB / ID: EMD-15378
TitleStructure of trimeric SlpA outer membrane protein
Map dataRELION PostProcessed map with B-factor sharpening
Sample
  • Complex: Structure of trimeric SlpA protein
    • Protein or peptide: S-layer protein SlpA
  • Ligand: CALCIUM IONCalcium
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 Å
Authorsvon Kuegelgen A / Bharat TAM
Funding support United Kingdom, 3 items
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, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateNov 30, 2022-
Current statusNov 30, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15378.png

Downloads & links

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Map

FileDownload / File: emd_15378.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION PostProcessed map with B-factor sharpening
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 320 pix.
= 349.44 Å		1.09 Å/pix.
x 320 pix.
= 349.44 Å		1.09 Å/pix.
x 320 pix.
= 349.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.092 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.052389175 - 0.09440462
Average (Standard dev.)1.370914e-05 (±0.0031721287)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 349.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15378_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map RELION without B-factor sharpening

Fileemd_15378_additional_1.map
AnnotationFull map RELION without B-factor sharpening
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION Half map 1

Fileemd_15378_half_map_1.map
AnnotationRELION Half map 1
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION Half map 1

Fileemd_15378_half_map_2.map
AnnotationRELION Half map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of trimeric SlpA protein

EntireName: Structure of trimeric SlpA protein
Components
  • Complex: Structure of trimeric SlpA protein
    • Protein or peptide: S-layer protein SlpA
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Structure of trimeric SlpA protein

SupramoleculeName: Structure of trimeric SlpA protein / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Structure of trimeric SlpA protein
Source (natural)Organism: Deinococcus radiodurans (radioresistant) / Strain: BAA-816

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Macromolecule #1: S-layer protein SlpA

MacromoleculeName: S-layer protein SlpA / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Deinococcus radiodurans (radioresistant)
Strain: ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422
Molecular weightTheoretical: 123.835367 KDa
SequenceString: MKKSLIALTT ALSFGLAAAQ TAAPVSAPQV PALTDVPAGH WAKDAIDRLV SRGVILGYPD GTFRGTQNLT RYEAAIIIAR LLDQMRDGE TPAGMTAEDM TALQNAIQEL AADLAALGVR VSDLEANAVS KDDFARLEAR IEEVAAAGGE QGATEALQGQ I DDLTARVD ...String:
MKKSLIALTT ALSFGLAAAQ TAAPVSAPQV PALTDVPAGH WAKDAIDRLV SRGVILGYPD GTFRGTQNLT RYEAAIIIAR LLDQMRDGE TPAGMTAEDM TALQNAIQEL AADLAALGVR VSDLEANAVS KDDFARLEAR IEEVAAAGGE QGATEALQGQ I DDLTARVD EYDALRADVD DNASSIAALN DLTVLLNQDI LDLQDRVSAV EAAQADFVQR SDFDALGGRV TTVETRVETV NN SLTGRIA ALERNAFSVK PSLTIGYSVS RTSRNFDVDR LFPLNADGTV ANNAFTSGGI DTDTGAQRRD FGDFGNASDP VVA GAAGLY GFADGVSYTV YFTDGSTATF DGLNPADYKV PTGKVIDTTK GRNGFGFNNL ARYKEGSTDI GISLGFDTSG QFSQ VTSGT GGSLFSTAGR LQVNQIDLNF GLVTGLPSDA YVDTNGNGKK DDGEATGRGT YLGSGGTAAI LRDPAGNVYR PVFFR FKNA TTQFSVGNNP VIVTLGQQQK FYFSDYVFDN NYDGRGDGFT VTVDGSNVPV IGAWKPQIKG VYGSRSGLDG TAEAGY GVY YRGVRAQITP VGTLTAGIHY AQEGRDMFGA AQNTTSTPSD VTTYGADLHG KAFGVELHSE YATSRVRPNT ANAAVQT SN AFYARVATRK DNLAFDLNTP AAKFGNDTFG VSLYDLNYRK IDAGYNNVAG ISEYGYGSYS RTSAQNIAYN PDTGVTAP F ANLDRQAYTD ANNDGTSDRN ADGTVVATNT KIGQMGFGVK AAANLGPVAI GGYYDTSTGA NGDNANRMTE AGGSAKVAY SIFSLRGTYN TLDSNRPQIY RDAAGTQIIG DAKVRRYAVQ ADVTPGLGLF VGAYYRDVNV NGVRSTTDRG LLGRGYLASS FEPGVGNNA YRTGLRCADN NFGTGTRDID GVGGVLNPAV NLDQSRTATC FTSYGVEAGH AGDNANALVK DLFFRVGYSR V YVPTTATA TTGDFSGSVT YGDARYDRKV GVANVRLAGS FSTTNTQLDS RPAGTRGAVG LIVRTDPLEN VPFRPQFNGQ VG YYTADNR VAAGNYNANA TKYGAGVVLN DFLLPQTKIG VRYDGYMAQN RQYTPFDGDG TQGYFSDANN NRRTNLNGVY VEG AYQDLI FSYGTYTLSQ KDLNGVEYGS GINNGQPARG QTFKISYKVN F

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 18 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration4.45 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium chloride
0.02 % w/vC24H46O11DDM

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.
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 20 seconds, 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
Details: Vitrobot options: Blot time 4.5 seconds, Blot force -10,1, Wait time 10 seconds, Drain time 0.5 seconds.
DetailsPurified SlpA protein after size-exclusion chromatography

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
DetailsEPU software with faster acquisition mode AFIS (Aberration Free Image Shift).
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: 2294 / Average exposure time: 4.8 sec. / Average electron dose: 47.909 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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.
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Software - details: CTFFIND4 was used as implemented in RELION 3.1
Details: RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included.
Startup modelType of model: NONE / Details: RELION de-novo
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Angle assignment was performed within RELION3.1
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.1)
Details: The reconstruction was further improved by Bayesian particle polishing, and a focused 3D-classification without refinement of the poses. The final output map was obtained from 122,412 ...Details: The reconstruction was further improved by Bayesian particle polishing, and a focused 3D-classification without refinement of the poses. The final output map was obtained from 122,412 particles, which was post-processed using a smooth mask focused on the trimeric OMBB including the first heptad of the coil coiled with a global resolution of 3.25 Angstrom according to the gold standard Fourier shell correlation criterion of 0.143.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Angle assignment was performed within RELION3.1
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 122412
DetailsMovies 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).

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 51.67 / Target criteria: Best Fit
Output model

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

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