PDB-7ptr: Structure of hexameric S-layer protein from Haloferax volcanii archaea

Basic information

• Entry: Database: PDB / ID: 7ptr
• Title: Structure of hexameric S-layer protein from Haloferax volcanii archaea
• Components: Cell surface glycoprotein
• Keywords: STRUCTURAL PROTEIN / S-layer csg

Function / homology

Function:

S-layer / cell wall organization / extracellular region / plasma membrane

Domain/homology:

Surface glycoprotein signal peptide / Major cell surface glycoprotein / PGF-CTERM archaeal protein-sorting signal / PGF-CTERM motif

Component:

beta-D-glucopyranose / Cell surface glycoprotein

• Biological species: Haloferax volcanii DS2 (archaea)
• Method: ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
• Authors: von Kuegelgen, A. / Bharat, T.A.M.

Funding support

United Kingdom, 3items

Organization	Grant number	Country
Wellcome Trust	202231/Z/16/Z
Other private	Vallee Scholarship	United Kingdom
Leverhulme Trust	Philip Leverhulme Prize

• Citation: Journal: Cell Rep / Year: 2021; Title: Complete atomic structure of a native archaeal cell surface.; Authors: Andriko von Kügelgen / Vikram Alva / Tanmay A M Bharat / ; Abstract: Many prokaryotic cells are covered by an ordered, proteinaceous, sheet-like structure called a surface layer (S-layer). S-layer proteins (SLPs) are usually the highest copy number macromolecules in prokaryotes, playing critical roles in cellular physiology such as blocking predators, scaffolding membranes, and facilitating environmental interactions. Using electron cryomicroscopy of two-dimensional sheets, we report the atomic structure of the S-layer from the archaeal model organism Haloferax volcanii. This S-layer consists of a hexagonal array of tightly interacting immunoglobulin-like domains, which are also found in SLPs across several classes of archaea. Cellular tomography reveal that the S-layer is nearly continuous on the cell surface, completed by pentameric defects in the hexagonal lattice. We further report the atomic structure of the SLP pentamer, which shows markedly different relative arrangements of SLP domains needed to complete the S-layer. Our structural data provide a framework for understanding cell surfaces of archaea at the atomic level.

History

Deposition	Sep 27, 2021	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Dec 15, 2021	Provider: repository / Type: Initial release

Assembly

Deposited unit

A: Cell surface glycoprotein

B: Cell surface glycoprotein

C: Cell surface glycoprotein

D: Cell surface glycoprotein

E: Cell surface glycoprotein

F: Cell surface glycoprotein

hetero molecules

Summary:

• 494 kDa, 6 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	494,498	42
Polymers	490,534	6
Non-polymers	3,964	36
Water	0

1

Summary:

• Idetical with deposited unit
• defined by author
• Evidence: microscopy

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555		1

Calculated values:

Buried area	20500 Å2
ΔGint	-65 kcal/mol
Surface area	188040 Å2

Components

#1: Protein

A B C D E F
Cell surface glycoprotein / S-layer glycoprotein

Details:

Mass: 81755.602 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Haloferax volcanii DS2 (archaea) / Plasmid details: Allers et al 2004 / References: UniProt: P25062

#2: Chemical

A-901 A-902 A-903 B-901 B-902 B-903 C-901 C-902 C-903 D-901 D-902 D-903 E-901 E-902 E-903 F-901 F-902 F-903
ChemComp-CA / CALCIUM ION

Details:

Mass: 40.078 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION

#3: Sugar

A-904 A-905 A-906 B-904 B-905 B-906 C-904 C-905 C-906 D-904 D-905 D-906 E-904 E-905 E-906 F-904 F-905 F-906
ChemComp-BGC / beta-D-glucopyranose / beta-D-glucose / D-glucose / glucose / Glucose

Details:

Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: C₆H₁₂O₆ / Feature type: SUBJECT OF INVESTIGATION

Identifier:

Identifier	Type	Program
DGlcpb	CONDENSED IUPAC CARBOHYDRATE SYMBOL	GMML 1.0
b-D-glucopyranose	COMMON NAME	GMML 1.0
b-D-Glcp	IUPAC CARBOHYDRATE SYMBOL	PDB-CARE 1.0
Glc	SNFG CARBOHYDRATE SYMBOL	GMML 1.0

• Has ligand of interest: Y

Experimental details

Experiment

• Experiment: Method: ELECTRON MICROSCOPY
• EM experiment: Aggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

Sample preparation

• Component: Name: Structure of hexameric S-layer protein csg / Type: COMPLEX / Details: Structure of hexameric S-layer protein csg / Entity ID: #1 / Source: NATURAL
• Molecular weight: Experimental value: NO
• Source (natural): Organism: Haloferax volcanii DS2 (archaea) / Cellular location: Cell surface
• Buffer solution: pH: 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 vacuum fold pump. The pH of the HEPES stock solution was adjusted with sodium hydroxide at 4 deg C. 15 mM Calcium chloride was added 15 minutes before vitrification.

Buffer component

ID	Conc.	Name	Formula	Buffer-ID
1	20 mM	HEPES	C8H18N2O4S	1
2	150 mM	magnesium chloride	MgCl2	1
3	15 mM	calcium chloride	CaCl2	1
4	0.65 % (w/v)	CHAPS detergent	C32H58N2O7S	1

• Specimen: Conc.: 3.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES; Details: Purified csg protein mixed with 15 mM CaCl2 after 15 minutes incubation.
• Specimen support: Details: 20 seconds, 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
• Vitrification: Instrument: 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

Electron microscopy imaging

• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company
• Microscopy: Model: FEI TITAN KRIOS; Details: EPU software with faster acquisition mode AFIS (Aberration Free Image Shift).
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
• Electron lens: Mode: BRIGHT FIELDBright-field microscopy / 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 holder: Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
• Image recording: Average exposure time: 3.4 sec. / Electron dose: 51.441 e/Ų / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 18468 ; Details: Images were collected in two sessions movie-mode and subjected to 3.4 seconds of exposure where a total dose of 49 or 51.441 e-/A2 was applied, and 40 frames were recorded per movie. A total of 18468 movies were collected in two sessions with the same microscope and settings.
• EM imaging optics: Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
• Image scans: Width: 5760 / Height: 4092

Processing

• Software: Name: PHENIX / Version: 1.19_4092: / Classification: refinement

EM software

ID	Name	Version	Category	Details
2	Topaz	0.2.5	particle selection	ResNet8 trained model
3	EPU		image acquisition
5	CTFFIND	4.1.13	CTF correction	CTFFIND4 was used as implemented in RELION 3.1
8	Coot	0.9.2-pre	model fitting
10	RELION	3.1	initial Euler assignment
11	RELION	3.1	final Euler assignment
12	RELION	3.1	classification
13	RELION	3.1	3D reconstruction
14	PHENIX	1.19-4092	model refinement

• Image processing: 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 correction: Details: 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 selection: Num. of particles selected: 10558369 ; Details: 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 (Zivanov et al., 2020). 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 pixel boxes and classified using reference-free 2D classification. Particle coordinates belonging to class averages centered at the hexameric axis were used to train TOPAZ (Bepler et al., 2019) in 5x downsampled micrographs with the neural network architecture ResNet8. For the final reconstruction, particles were picked using TOPAZ and the previously trained neural network above. Additionally, top and bottom views were picked using the reference-based autopicker inside RELION3.1, which were not readily identified by TOPAZ. Particles were extracted in 4x downsampled 100 x 100 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 100 angstrom were removed to prevent duplication after alignment.
• Symmetry: Point symmetry: C₆ (6 fold cyclic)
• 3D reconstruction: Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1087798 / Algorithm: FOURIER SPACE; Details: Particles from classes with the same curvature were combined, re-extracted in 400 x 400 boxes and subjected to a focused 3D auto refinement on the central 6 subunits using the scaled and lowpass filtered output from the 3D classification as a starting model. 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 (Zivanov et al., 2020).; Num. of class averages: 1 / Symmetry type: POINT
• Atomic model building: B value: 143.26 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Best Fit; Details: The boundaries of the six Ig-like domains, D1-D6, were predicted using HHpred (Steinegger et al., 2019) in default settings within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018). Subsequently, structural models for these domains were built using the Robetta structure prediction server, employing the deep learning-based modelling method TrRosetta (Yang et al., 2020). The obtained structural models of domains D3-D6 resulted in an overall fit into the hexameric cryo-EM map of csg from the reconstituted sheets. D1-D2 deviated significantly from any obtained homology models, and for those domains, the carbon backbone of the csg protein was manually traced through a single subunit of the hexameric cryo-EM density using Coot (Emsley and Cowtan, 2004). Due to the edge effect of the box used in the refinement of the 3.5 angstrom map, parts of D6 displayed edge artefacts. These artefacts were removed using single-particle cryo-EM refinement in a larger box, which led to an overall slightly lower resolution (3.8 angstrom) but allowed fitting of the D6 homology model unambiguously. Following initial manual building (for D1-D2) or fitting in of structural models (for D3-D6), side chains were assigned in regions with density corresponding to characteristic aromatic residues allowing us to deduce the register of the amino acid sequence in the map. Another important check of the model building was the position of known glycan positions, which were readily assigned based on large unexplained densities on characteristic asparagine residues. The atomic model was then placed into the hexameric map in six copies and subjected to several rounds of refinement using refmac5 (Murshudov et al., 2011) inside the CCP-EM software suite (Burnley et al., 2017) and PHENIX (Liebschner et al., 2019), followed by manually rebuilding in Coot (Emsley and Cowtan, 2004). Model validation was performed in PHENIX and CCP-EM.