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- PDB-6t72: Structure of the RsaA N-terminal domain bound to LPS -

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Basic information

Entry
Database: PDB / ID: 6t72
TitleStructure of the RsaA N-terminal domain bound to LPS
ComponentsS-layer protein
KeywordsSTRUCTURAL PROTEIN / S-layer LPS RsaA
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 CB15 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
Authorsvon Kuegelgen, A. / Bharat, T.A.M.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
CitationJournal: 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.
History
DepositionOct 21, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2020Group: Data collection / Database references / Source and taxonomy
Category: chem_comp / citation / entity_src_gen
Item: _chem_comp.type / _citation.journal_volume ..._chem_comp.type / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation.year / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_host_org_scientific_name
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.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _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

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Structure visualization

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Assembly

Deposited unit
A: S-layer protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,1055
Polymers25,8201
Non-polymers2,2844
Water0
1
A: S-layer protein
hetero molecules
x 14


  • defined by author
  • Evidence: mass spectrometry, The complex was intensively studied with native mass spectrometry. Using high energy tandem mass spectrometry the stochiometry of the complex was determined as a mixture ...Evidence: mass spectrometry, The complex was intensively studied with native mass spectrometry. Using high energy tandem mass spectrometry the stochiometry of the complex was determined as a mixture of 20 and 21 subunits of the N-terminal domain of RsaA, 6 full lipopolysaccharide molecules and 1 copy of hydrolysed polysaccharide. Submitted structure is a focussed refinement on the central 14 subunits.
  • 393 kDa, 14 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)393,46670
Polymers361,48514
Non-polymers31,98156
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation13
Buried area1250 Å2
ΔGint-20 kcal/mol
Surface area12300 Å2

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Components

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


Mass: 25820.354 Da / Num. of mol.: 1 / Mutation: TEV site added at the position 250
Source method: isolated from a genetically manipulated source
Details: LPS O-antigen bound to protein / Source: (gene. exp.) Caulobacter vibrioides CB15 (bacteria) / Gene: rsaA, CC_1007 / Production host: Caulobacter vibrioides CB15 (bacteria) / 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-(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: 2164.121 Da / Num. of mol.: 1 / Source method: obtained synthetically
DescriptorTypeProgram
WURCS=2.0/2,12,11/[a1122h-1b_1-5][a1122m-1a_1-5_4*NCC/3=O]/1-2-2-1-2-2-1-2-2-1-2-2/a3-b1_b3-c1_c3-d1_d3-e1_e3-f1_f3-g1_g3-h1_h3-i1_i3-j1_j3-k1_k3-l1WURCSPDB2Glycan 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]{[(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.: 3 / 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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structure of RsaA N-terminal domain bound to LPS / Type: COMPLEX / Details: Structure of RsaA N-terminal domain bound to LPS / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.577 MDa / Experimental value: YES
Source (natural)Organism: Caulobacter vibrioides CB15 (bacteria) / Strain: YB1001
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
125 mMHEPESC8H18N2O4S1
2100 mMsodium chlorideNaClSodium chloride1
31 mMmagnesium chlorideMgCl21
41 mMcalcium chlorideCaCl21
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: RsaA N-terminal domain with LPS
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 3 seconds, Blot force -13,1, 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: FEI TITAN KRIOS / Details: EPU software
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 130000 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: 8 sec. / Electron dose: 43 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2422
Details: Images were collected in movie-mode and subjected to 8 seconds of exposure where a total dose of 43 e-/A2 was applied, and 20 frames were recorded per movie.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 20 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selectionRELION was used throughout the entire single particle analysis.
2EPUimage acquisition
4CTFFIND4.1.13CTF correctionCTFFIND was used as implemented in Relion 3.0
7Coot0.9-premodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13REFMAC5model refinement
Image processingDetails: Movies were motion corrected and dose weighted with MotionCor2 (Zheng et al., 2017) implemented in Relion 3.0 (Zivanov et al., 2018). Contrast transfer functions (CTFs) of the resulting ...Details: Movies were motion corrected and dose weighted with MotionCor2 (Zheng et al., 2017) implemented in Relion 3.0 (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: 129633
Details: Particles were automatically picked from the motion and CTF corrected micrographs using the AutoPick function in Relion 3.0 (Zivanov et al., 2018). As particle reference a 3 dimensional ...Details: Particles were automatically picked from the motion and CTF corrected micrographs using the AutoPick function in Relion 3.0 (Zivanov et al., 2018). As particle reference a 3 dimensional reconstruction from an earlier dataset with different pixelsize was used which was reconstructed using an unbiased subtomogram average structure of the same sample.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115776 / Algorithm: FOURIER SPACE
Details: Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using the output from the 3D classification as ...Details: Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using the output from the 3D classification as a starting model. The final map was obtained from 115,776 particles and post-processed using a soft mask focused on the inner fourteen subunits.
Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 85.819 / Protocol: BACKBONE TRACE / Space: RECIPROCAL / Target criteria: Best fit
Details: The carbon backbone of the RsaA protein was manually traced through a single subunit of the cryo-EM density using Coot (Emsley et al., 2010). Initially, side chains were assigned in regions ...Details: The carbon backbone of the RsaA protein was manually traced through a single subunit of the cryo-EM density using Coot (Emsley et al., 2010). Initially, 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. Side chains for residues 2-243 of RsaA were thus assigned unambiguously and the structure was refined and manually rebuilt using Refmac5 (Murshudov et al., 2011) inside the CCP-EM (Burnley et al., 2017) software suite and Coot.

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