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- PDB-6z7p: Composite model of the Caulobacter crescentus S-layer bound to th... -

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Basic information

Entry
Database: PDB / ID: 6z7p
TitleComposite model of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide
ComponentsS-layer protein
KeywordsSTRUCTURAL PROTEIN / RsaA S-layer sub-tomogram averaging Caulobacter lipopolysaccharide O-antigen
Function / homologyRsaA N-terminal domain / S-layer / RTX calcium-binding nonapeptide repeat / RTX calcium-binding nonapeptide repeat (4 copies) / Serralysin-like metalloprotease, C-terminal / calcium ion binding / extracellular region / S-layer protein
Function and homology information
Biological speciesCaulobacter vibrioides (bacteria)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 4.8 Å
AuthorsBharat, T.A.M. / von Kugelgen, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Citation
Journal: Cell / Year: 2020
Title: In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.
Authors: Andriko von Kügelgen / Haiping Tang / Gail G Hardy / Danguole Kureisaite-Ciziene / Yves V Brun / Phillip J Stansfeld / Carol V Robinson / Tanmay A M Bharat /
Abstract: Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are ...Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.
#1: Journal: Nat Microbiol / Year: 2017
Title: Structure of the hexagonal surface layer on Caulobacter crescentus cells.
Authors: Tanmay A M Bharat / Danguole Kureisaite-Ciziene / Gail G Hardy / Ellen W Yu / Jessica M Devant / Wim J H Hagen / Yves V Brun / John A G Briggs / Jan Löwe /
Abstract: Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and ...Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and Gram-negative bacteria and most archaea. S-layers protect cells from the outside, provide mechanical stability and also play roles in pathogenicity. In situ structural information about this highly abundant class of proteins is scarce, so atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified Caulobacter crescentus' sole S-layer protein RsaA, we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. We also solved a 7.4 Å structure of the S-layer through electron cryotomography and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the electron cryotomography map, resulting in a pseudo-atomic-level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. The cellular S-layer atomic structure shows that the S-layer is porous, with a largest gap dimension of 27 Å, and is stabilized by multiple Ca ions bound near the interfaces. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with electron cryotomography and sub-nanometre-resolution sub-tomogram averaging.
History
DepositionJun 1, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 15, 2020Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / struct_asym / struct_conn / struct_conn_type / struct_site / struct_site_gen
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1May 15, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Assembly

Deposited unit
A: S-layer protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,53224
Polymers98,0231
Non-polymers2,50923
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: microscopy, The assembly of the complex has been observed using cryo-electron microscopy and cryo-electron tomography.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1910 Å2
ΔGint-137 kcal/mol
Surface area40960 Å2
MethodPISA

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Components

#1: Protein S-layer protein / / Paracrystalline surface layer protein


Mass: 98022.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Caulobacter vibrioides (strain ATCC 19089 / CB15) (bacteria)
Strain: ATCC 19089 / CB15 / References: UniProt: P35828
#2: Polysaccharide 4-acetamido-4,6-dideoxy-alpha-D-mannopyranose-(1-3)-4-acetamido-4,6-dideoxy-alpha-D-mannopyranose- ...4-acetamido-4,6-dideoxy-alpha-D-mannopyranose-(1-3)-4-acetamido-4,6-dideoxy-alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-3)-4-acetamido-4,6-dideoxy-alpha-D-mannopyranose-(1-3)-4-acetamido-4,6-dideoxy-alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-3)-4-acetamido-4,6-dideoxy-alpha-D-mannopyranose-(1-3)-4-acetamido-4,6-dideoxy-alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose


Type: oligosaccharide / Mass: 1627.594 Da / Num. of mol.: 1 / Source method: obtained synthetically
DescriptorTypeProgram
WURCS=2.0/2,9,8/[a1122h-1b_1-5][a1122m-1a_1-5_4*NCC/3=O]/1-2-2-1-2-2-1-2-2/a3-b1_b3-c1_c3-d1_d3-e1_e3-f1_f3-g1_g3-h1_h3-i1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-Manp]{[(3+1)][a-D-Rhap4NAc]{[(3+1)][a-D-Rhap4NAc]{[(3+1)][b-D-Manp]{[(3+1)][a-D-Rhap4NAc]{[(3+1)][a-D-Rhap4NAc]{[(3+1)][b-D-Manp]{[(3+1)][a-D-Rhap4NAc]{[(3+1)][a-D-Rhap4NAc]{}}}}}}}}}LINUCSPDB-CARE
#3: Chemical...
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 22 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: S-layer / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Caulobacter vibrioides NA1000 (bacteria) / Strain: YB2811 / Cellular location: extra-cellular
Buffer solutionpH: 7 / Details: PYE
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Caulobacter crescentus stalk
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: 1.5 s blot

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 105000 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: 1 sec. / Electron dose: 3.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Details: Dose symmetric tilt scheme (Hagen et al, JSB)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Chromatic aberration corrector: not used / Energyfilter slit width: 20 eV / Spherical aberration corrector: not used

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Processing

EM software
IDNameVersionCategoryDetails
1RELION1.4volume selection
2SerialEMimage acquisition
7UCSF Chimera1.13model fittingRigid body fit
8Coot0.9-premodel fittingRigid body fit
CTF correctionDetails: Following Turonova and Briggs NovaCTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51866 / Algorithm: FOURIER SPACE
Details: Tomogram generation in IMOD using the NovaCTF implementation of Turonova and Briggs. Sub-tomogram averaging performed using the AV3 package (Friedrich Foerster) applied to tubular specimens ...Details: Tomogram generation in IMOD using the NovaCTF implementation of Turonova and Briggs. Sub-tomogram averaging performed using the AV3 package (Friedrich Foerster) applied to tubular specimens (Bharat et al, PLoS Biology, 2011)
Num. of class averages: 1 / Symmetry type: POINT
EM volume selectionMethod: RELIONList of Walmart brands / Details: RELION subtomogram averaging / Num. of tomograms: 110 / Num. of volumes extracted: 51866 / Reference model: Ab initio
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: USCF Chimera
Details: Initial rigid body fit of the C-terminal X-ray structure (PDB: 5N8P, amino acids 249-1046) and the N-terminal cryo-EM structure (PDB: 6T72, amino acids 2-243) was performed in USCF Chimera. ...Details: Initial rigid body fit of the C-terminal X-ray structure (PDB: 5N8P, amino acids 249-1046) and the N-terminal cryo-EM structure (PDB: 6T72, amino acids 2-243) was performed in USCF Chimera. The missing amino acid linker was manually added suing Coot. The model was not refined against the map and all B-factors were set to zero due to resolution anisotropy.
Atomic model building

3D fitting-ID: 1 / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-IDPdb chain residue range
15N8P

5n8p

5N8P1249-1046
26T72

6t72

6T7222-243

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