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- EMDB-16183: In situ structure of the Caulobacter crescentus S-layer -

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Basic information

Entry
Database: EMDB / ID: EMD-16183
TitleIn situ structure of the Caulobacter crescentus S-layer
Map dataPostProcessed map with B-factor sharpening
Sample
  • Organelle or cellular component: Caulobacter crescentus S-layer
    • Protein or peptide: S-layer protein rsaA
  • Ligand: CALCIUM IONCalcium
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)
Methodsubtomogram averaging / cryo EM / Resolution: 3.5 Å
Authorsvon Kuegelgen A / Bharat T
Funding support United Kingdom, 2 items
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 TAM / Briggs JAG / Scheres SHW
History
DepositionNov 21, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateDec 28, 2022-
Current statusDec 28, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16183.png

Downloads & links

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Map

FileDownload / File: emd_16183.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostProcessed map with B-factor sharpening
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 160 pix.
= 216. Å		1.35 Å/pix.
x 160 pix.
= 216. Å		1.35 Å/pix.
x 160 pix.
= 216. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.54716
Minimum - Maximum-1.4492271 - 2.1447482
Average (Standard dev.)1.8384515e-12 (±0.109431066)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 216.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16183_msk_1.map
Projections & Slices
AxesZYX

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Half map: Half map 1

Fileemd_16183_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_16183_half_map_2.map
AnnotationHalf map 2
Projections & Slices
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Sample components

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Entire : Caulobacter crescentus S-layer

EntireName: Caulobacter crescentus S-layer
Components
  • Organelle or cellular component: Caulobacter crescentus S-layer
    • Protein or peptide: S-layer protein rsaA
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Caulobacter crescentus S-layer

SupramoleculeName: Caulobacter crescentus S-layer / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Caulobacter crescentus S-layer
Source (natural)Organism: Caulobacter vibrioides NA1000 (bacteria) / Strain: YB2811 / Location in cell: extra-cellular

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Macromolecule #1: S-layer protein rsaA

MacromoleculeName: S-layer protein rsaA / type: protein_or_peptide / ID: 1 / Details: In-situ S-layer / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Caulobacter vibrioides NA1000 (bacteria) / Strain: NA1000 / CB15N
Molecular weightTheoretical: 98.153906 KDa
Recombinant expressionOrganism: Caulobacter vibrioides NA1000 (bacteria)
SequenceString: MAYTTAQLVT AYTNANLGKA PDAATTLTLD AYATQTQTGG LSDAAALTNT LKLVNSTTAV AIQTYQFFTG VAPSAAGLDF LVDSTTNTN DLNDAYYSKF AQENRFINFS INLATGAGAG ATAFAAAYTG VSYAQTVATA YDKIIGNAVA TAAGVDVAAA V AFLSRQAN ...String:
MAYTTAQLVT AYTNANLGKA PDAATTLTLD AYATQTQTGG LSDAAALTNT LKLVNSTTAV AIQTYQFFTG VAPSAAGLDF LVDSTTNTN DLNDAYYSKF AQENRFINFS INLATGAGAG ATAFAAAYTG VSYAQTVATA YDKIIGNAVA TAAGVDVAAA V AFLSRQAN IDYLTAFVRA NTPFTAAADI DLAVKAALIG TILNAATVSG IGGYATATAA MINDLSDGAL STDNAAGVNL FT AYPSSGV SGSTLSLTTG TDTLTGTANN DTFVAGEVAG AATLTVGDTL SGGAGTDVLN WVQAAAVTAL PTGVTISGIE TMN VTSGAA ITLNTSSGVT GLTALNTNTS GAAQTVTAGA GQNLTATTAA QAANNVAVDG GANVTVASTG VTSGTTTVGA NSAA SGTVS VSVANSSTTT TGAIAVTGGT AVTVAQTAGN AVNTTLTQAD VTVTGNSSTT AVTVTQTAAA TAGATVAGRV NGAVT ITDS AAASATTAGK IATVTLGSFG AATIDSSALT TVNLSGTGTS LGIGRGALTA TPTANTLTLN VNGLTTTGAI TDSEAA ADD GFTTINIAGS TASSTIASLV AADATTLNIS GDARVTITSH TAAALTGITV TNSVGATLGA ELATGLVFTG GAGADSI LL GATTKAIVMG AGDDTVTVSS ATLGAGGSVN GGDGTDVLVA NVNGSSFSAD PAFGGFETLR VAGAAAQGSH NANGFTAL Q LGATAGATTF TNVAVNVGLT VLAAPTGTTT VTLANATGTS DVFNLTLSSS AALAAGTVAL AGVETVNIAA TDTNTTAHV DTLTLQATSA KSIVVTGNAG LNLTNTGNTA VTSFDASAVT GTGSAVTFVS ANTTVGEVVT IRGGAGADSL TGSATANDTI IGGAGADTL VYTGGTDTFT GGTGADIFDI NAIGTSTAFV TITDAAVGDK LDLVGISTNG AIADGAFGAA VTLGAAATLA Q YLDAAAAG DGSGTSVAKW FQFGGDTYVV VDSSAGATFV SGADAVIKLT GLVTLTTSAF ATEVLTLA

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / 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
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7 / Details: PYE medium
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV / Details: 1.5 s blot.
DetailsCaulobacter crescentus stalk

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 105000
Specialist opticsSpherical aberration corrector: not used / Chromatic aberration corrector: not used / Energy filter - Name: GIF Quantum LS / 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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-10 / Number real images: 1 / Average exposure time: 1.0 sec. / Average electron dose: 3.4 e/Å2 / Details: Dose symmetric tilt scheme (Hagen et al, JSB)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 110 / Number images used: 51866 / Reference model: EMDF / Method: EMD-10388 / Software - Name: RELION (ver. 4.0.0) / Details: RELION subtomogram averaging
Final 3D classificationNumber classes: 6 / Avg.num./class: 8645 / Software - Name: RELION (ver. 4.0.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0.0) / Details: RELION 4.0.0
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 42990

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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