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- EMDB-15399: Cryo-EM map of crescentin filament in complex with a megabody (st... -

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Basic information

Entry
Database: EMDB / ID: EMD-15399
TitleCryo-EM map of crescentin filament in complex with a megabody (stutter mutant, C2 symmetry, large box)
Map data
Sample
  • Complex: Crescentin filament in complex with megabody MB13
    • Complex: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)
    • Complex: Crescentin-specific megabody MB13
    • Protein or peptide: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)
    • Protein or peptide: Crescentin-specific megabody MB13
Keywordscytoskeleton / cell shape / intermediate filaments / coiled coil / assembly / STRUCTURAL PROTEIN
Biological speciesCaulobacter vibrioides (bacteria) / Camelidae (mammal)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsLiu Y / Lowe J
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust202754/Z/16/Z United Kingdom
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2024
Title: Filament structure and subcellular organization of the bacterial intermediate filament-like protein crescentin.
Authors: Yue Liu / Fusinita van den Ent / Jan Löwe /
Abstract: The protein crescentin is required for the crescent shape of the freshwater bacterium (). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features ...The protein crescentin is required for the crescent shape of the freshwater bacterium (). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, including the formation of static filaments based on long and parallel coiled coils, the protein's length, structural roles in cell and organelle shape determination and the presence of a coiled coil discontinuity called the "stutter." Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by twofold symmetry, that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine cross-linking, we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. When comparing with current models of IF proteins and their filaments, which are also built from parallel coiled coil dimers and lack overall polarity, it emerges that IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures.
#1: Journal: to be published
Title: Assembly and organization of the bacterial intermediate filament-like protein crescentin
Authors: Liu Y / Lowe J
History
DepositionJul 18, 2022-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15399.png

Downloads & links

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Map

FileDownload / File: emd_15399.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.59 Å
Density
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.002505131 - 3.2213197
Average (Standard dev.)0.00013104687 (±0.0076462543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 954.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15399_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15399_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15399_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Crescentin filament in complex with megabody MB13

EntireName: Crescentin filament in complex with megabody MB13
Components
  • Complex: Crescentin filament in complex with megabody MB13
    • Complex: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)
    • Complex: Crescentin-specific megabody MB13
    • Protein or peptide: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)
    • Protein or peptide: Crescentin-specific megabody MB13

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Supramolecule #1: Crescentin filament in complex with megabody MB13

SupramoleculeName: Crescentin filament in complex with megabody MB13 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)

SupramoleculeName: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)
type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Caulobacter vibrioides (bacteria)

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Supramolecule #3: Crescentin-specific megabody MB13

SupramoleculeName: Crescentin-specific megabody MB13 / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Camelidae (mammal)

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Macromolecule #1: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)

MacromoleculeName: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Caulobacter vibrioides (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRLLSKNSRE TKNGKPTVLG DEARAEAMQH QIESTQAIGQ RYETIHGGLD SIGRVMEHLK AIEPLIAEIR GPVSQEFEAR RAEHAELIAV RANLDQAQRQ IALIQAEERE VSARLAAAET ALGESDARRQ TQDAALEDNA LEIDRLRNAL LQSDLKVSSL DASLRDATAR ...String:
MRLLSKNSRE TKNGKPTVLG DEARAEAMQH QIESTQAIGQ RYETIHGGLD SIGRVMEHLK AIEPLIAEIR GPVSQEFEAR RAEHAELIAV RANLDQAQRQ IALIQAEERE VSARLAAAET ALGESDARRQ TQDAALEDNA LEIDRLRNAL LQSDLKVSSL DASLRDATAR IEHLVQDVEG LRVQAQDIDA RRGDAEAALA RANQDNALLG EEAATLKKRV DQAGLDLARL SRIETDLEAQ LAAERARVQA VENALAAHQA DSGRTIRGLE SQVEANRAEI SALQTRLETA TGRADKLEEM NGQISARLAD SSAQQKAVER RAGDLNVALE RALDRIRALE EEADGLRQRH AGVDTARATA IERADQLAKS AVAQEKALKR AEERAQQLRA RLDAMQEAQD QVRRDSATHE AKIAELQATI ERLTSEAALA EGALEAARRD RSRLQMALLG ASDGDVAASA

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Macromolecule #2: Crescentin-specific megabody MB13

MacromoleculeName: Crescentin-specific megabody MB13 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Camelidae (mammal)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EVQLQESGGG LVYKEETQSG LNNYARVVEK GQYDSLEIPA QVAASWESGR DDAAVFGFID KEQLDKYVAN GGKRSDWTVK FAENRSQDGT LLGYSLLQES VDQASYMYSD NHYLAEMATI LGKPEEAKRY RQLAQQLADY INTCMFDPTT QFYYDVRIED KPLANGCAGK ...String:
EVQLQESGGG LVYKEETQSG LNNYARVVEK GQYDSLEIPA QVAASWESGR DDAAVFGFID KEQLDKYVAN GGKRSDWTVK FAENRSQDGT LLGYSLLQES VDQASYMYSD NHYLAEMATI LGKPEEAKRY RQLAQQLADY INTCMFDPTT QFYYDVRIED KPLANGCAGK PIVERGKGPE GWSPLFNGAA TQANADAVVK VMLDPKEFNT FVPLGTAALT NPAFGADIYW RGRVWVDQFW FGLKGMERYG YRDDALKLAD TFFRHAKGLT ADGPIQENYN PLTGAQQGAP NFSWSAAHLY MLYNDFFRKQ ASGGGSGGGG SGGGGSGNAD NYKNVINRTG APQYMKDYDY DDHQRFNPFF DLGAWHGHLL PDGPNTMGGF PGVALLTEEY INFMASNFDR LTVWQDGKKV DFTLEAYSIP GALVQKLTAK DVQVEMTLRF ATPRTSLLET KITSNKPLDL VWDGELLEKL EAKEGKPLSD KTIAGEYPDY QRKISATRDG LKVTFGKVRA TWDLLTSGES EYQVHKSLPV QTEINGNRFT SKAHINGSTT LYTTYSHLLT AQEVSKEQMQ IRDILARPAF YLTASQQRWE EYLKKGLTNP DATPEQTRVA VKAIETLNGN WRSPGGAVKF NTVTPSVTGR WFSGNQTWPW DTWKQAFAMA HFNPDIAKEN IRAVFSWQIQ PGDSVRPQDV GFVPDLIAWN LSPERGGDGG NWNERNTKPS LAAWSVMEVY NVTQDKTWVA EMYPKLVAYH DWWLRNRDHN GNGVPEYGAT RDKAHNTESG EMLFTVKKDS LRLSCASSRS IDGINIMRWY RQAPGKQRGM VAVVTGWGST NYVDSVKGRF IISRDSAKDT VYLQMNNLKP EDTAVYSCNA IYRGSEYWGQ GTQVTVSSGE NLYFQGSHHH HHHHHHH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration2 mg/mL
BufferpH: 6.5 / Details: PIPES 25mM pH 6.5, 0.05% CHAPS
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Average exposure time: 2.4 sec. / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software: (Name: cryoSPARC, RELION)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 585252
FSC plot (resolution estimation)

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