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- PDB-8bqe: In situ structure of the Caulobacter crescentus S-layer -

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Basic information

Entry
Database: PDB / ID: 8bqe
TitleIn situ structure of the Caulobacter crescentus S-layer
ComponentsS-layer protein rsaA
KeywordsSTRUCTURAL PROTEIN / RsaA S-layer sub-tomogram averaging Caulobacter
Function / homologyRsaA N-terminal domain / RTX calcium-binding nonapeptide repeat / RTX calcium-binding nonapeptide repeat (4 copies) / Serralysin-like metalloprotease, C-terminal / calcium ion binding / S-layer protein rsaA
Function and homology information
Biological speciesCaulobacter vibrioides NA1000 (bacteria)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 3.5 Å
Authorsvon Kuegelgen, A. / Bharat, T.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/31 United Kingdom
Citation
Journal: Elife / Year: 2022
Title: A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0.
Authors: Jasenko Zivanov / Joaquín Otón / Zunlong Ke / Andriko von Kügelgen / Euan Pyle / Kun Qu / Dustin Morado / Daniel Castaño-Díez / Giulia Zanetti / Tanmay A M Bharat / John A G Briggs / Sjors H W Scheres /
Abstract: We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are ...We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe the approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt-series alignments, beam-induced motions of the particles throughout the tilt-series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, particularly for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.
#1: Journal: BioRxiv / Year: 2022
Title: A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0
Authors: Zivanov, J. / Oton, J. / Ke, Z. / von Kuegelgen, A. / Pyle, E. / Qu, K. / Morado, D. / Castano-Diez, D. / Zanetti, G. / Bharat, T.A.M. / Briggs, J.A.G. / Scheres, S.H.W.
History
DepositionNov 21, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: S-layer protein rsaA
C: S-layer protein rsaA
D: S-layer protein rsaA
E: S-layer protein rsaA
F: S-layer protein rsaA
A: S-layer protein rsaA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)602,73836
Polymers588,9236
Non-polymers13,81430
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
S-layer protein rsaA


Mass: 98153.906 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: In-situ S-layer
Source: (gene. exp.) Caulobacter vibrioides NA1000 (bacteria)
Strain: NA1000 / CB15N / Gene: rsaA, CCNA_01059 / Production host: Caulobacter vibrioides NA1000 (bacteria) / Strain (production host): YB2811 / References: UniProt: A0A0H3C8J1
#2: Polysaccharide
4-acetamido-4,6-dideoxy-alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-3)-4-acetamido-4,6- ...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


Type: oligosaccharide / Mass: 1091.068 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
WURCS=2.0/2,6,5/[a1122m-1a_1-5_4*NCC/3=O][a1122h-1b_1-5]/1-2-1-1-2-1/a3-b1_b3-c1_c3-d1_d3-e1_e3-f1WURCSPDB2Glycan 1.1.0
[][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]{}}}}}}LINUCSPDB-CARE
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 18 / 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: Caulobacter crescentus S-layer / Type: ORGANELLE OR CELLULAR COMPONENT / Details: Caulobacter crescentus S-layer / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Caulobacter vibrioides NA1000 (bacteria) / Strain: YB2811 / Cellular location: extra-cellular
Buffer solutionpH: 7 / Details: PYE medium
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: ETHANE / 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 FIELD / 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 / Avg electron dose per subtomogram: 140 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 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
Image scansMovie frames/image: 10 / Used frames/image: 1-10

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
1RELION4.0.0volume selection
2SerialEMimage acquisition
4RELION4.0.0CTF correction
7PHENIX1.19-4092model fitting
9PHENIX1.19-4092model refinement
11RELION4.0.0final Euler assignment
12RELION4.0.0classification
13RELION3D reconstruction
CTF correctionDetails: PseudoSubtomograms as described in Zivanov 2022 (https://www.biorxiv.org/content/10.1101/2022.02.28.482229v2.abstract)
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42990 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
EM volume selectionMethod: EMD-10388 / Details: RELION subtomogram averaging / Num. of tomograms: 110 / Num. of volumes extracted: 51866 / Reference model: EMDF
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00111412
ELECTRON MICROSCOPYf_angle_d0.28115690
ELECTRON MICROSCOPYf_dihedral_angle_d3.0632088
ELECTRON MICROSCOPYf_chiral_restr0.0312130
ELECTRON MICROSCOPYf_plane_restr0.0011920

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