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- EMDB-15476: Cryo-EM structure of crescentin filaments (stutter mutant, C2 sym... -

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Basic information

Entry
Database: EMDB / ID: EMD-15476
TitleCryo-EM structure of crescentin filaments (stutter mutant, C2 symmetry and large box)
Map data
Sample
  • Complex: Crescentin filament in complex with megabody MB13
    • Complex: Crescentin
      • Protein or peptide: Crescentin
    • Complex: Crescentin-specific megabody MB13
      • Protein or peptide: Crescentin-specific megabody MB13
Keywordscytoskeleton / cell shape / intermediate filaments / coiled coil / assembly / STRUCTURAL PROTEIN
Function / homology:
Function and homology information
Biological speciesCaulobacter vibrioides (bacteria) / Camelidae (mammal)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 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 28, 2022-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15476.png

Downloads & links

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Map

FileDownload / File: emd_15476.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.59 Å/pix.
x 600 pix.
= 954. Å		1.59 Å/pix.
x 600 pix.
= 954. Å		1.59 Å/pix.
x 600 pix.
= 954. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.59 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.022
Minimum - Maximum-0.0017254615 - 2.736003
Average (Standard dev.)0.000074802236 (±0.0063851704)
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_15476_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_15476_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_15476_half_map_2.map
Projections & Slices
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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: Crescentin
      • Protein or peptide: Crescentin
    • Complex: Crescentin-specific megabody MB13
      • 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: Crescentin

SupramoleculeName: Crescentin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #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 / Macromolecule list: #2
Source (natural)Organism: Camelidae (mammal)

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Macromolecule #1: Crescentin

MacromoleculeName: Crescentin / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Caulobacter vibrioides (bacteria)
Molecular weightTheoretical: 50.177816 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRLLSKNSRE TKNGKPTVLG DEARAEAMQH QIESTQAIGQ RYETIHGGLD SIGRVMEHLK AIEPLIAEIR GPVSQEFEAR RAEHAELIA VRANLDQAQR QIALIQAEER EVSARLAAAE TALGESDARR QTQDAALEDN ALEIDRLRNA LLQSDLKVSS L DASLRDAT ...String:
MRLLSKNSRE TKNGKPTVLG DEARAEAMQH QIESTQAIGQ RYETIHGGLD SIGRVMEHLK AIEPLIAEIR GPVSQEFEAR RAEHAELIA VRANLDQAQR QIALIQAEER EVSARLAAAE TALGESDARR QTQDAALEDN ALEIDRLRNA LLQSDLKVSS L DASLRDAT ARIEHLVQDV EGLRVQAQDI DARRGDAEAA LARANQDNAL LGEEAATLKK RVDQAGLDLA RLSRIETDLE AQ LAAERAR VQAVENALAA HQADSGRTIR GLESQVEANR AEISALQTRL ETATGRADKL EEMNGQISAR LADSSAQQKA VER RAGDLN VALERALDRI RALEEEADGL RQRHAGVDTA RATAIERADQ LAKSAVAQEK ALKRAEERAQ QLRARLDAMQ EAQD QVRRD SATHEAKIAE LQATIERLTS EAALAEGALE AARRDRSRLQ MALLGASDGD VAASA

UniProtKB: UNIPROTKB: A0A8F8EC09

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Macromolecule #2: Crescentin-specific megabody MB13

MacromoleculeName: Crescentin-specific megabody MB13 / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Camelidae (mammal)
Molecular weightTheoretical: 101.79018 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: EVQLQESGGG LVYKEETQSG LNNYARVVEK GQYDSLEIPA QVAASWESGR DDAAVFGFID KEQLDKYVAN GGKRSDWTVK FAENRSQDG TLLGYSLLQE SVDQASYMYS DNHYLAEMAT ILGKPEEAKR YRQLAQQLAD YINTCMFDPT TQFYYDVRIE D KPLANGCA ...String:
EVQLQESGGG LVYKEETQSG LNNYARVVEK GQYDSLEIPA QVAASWESGR DDAAVFGFID KEQLDKYVAN GGKRSDWTVK FAENRSQDG TLLGYSLLQE SVDQASYMYS DNHYLAEMAT ILGKPEEAKR YRQLAQQLAD YINTCMFDPT TQFYYDVRIE D KPLANGCA GKPIVERGKG PEGWSPLFNG AATQANADAV VKVMLDPKEF NTFVPLGTAA LTNPAFGADI YWRGRVWVDQ FW FGLKGME RYGYRDDALK LADTFFRHAK GLTADGPIQE NYNPLTGAQQ GAPNFSWSAA HLYMLYNDFF RKQASGGGSG GGG SGGGGS GNADNYKNVI NRTGAPQYMK DYDYDDHQRF NPFFDLGAWH GHLLPDGPNT MGGFPGVALL TEEYINFMAS NFDR LTVWQ DGKKVDFTLE AYSIPGALVQ KLTAKDVQVE MTLRFATPRT SLLETKITSN KPLDLVWDGE LLEKLEAKEG KPLSD KTIA GEYPDYQRKI SATRDGLKVT FGKVRATWDL LTSGESEYQV HKSLPVQTEI NGNRFTSKAH INGSTTLYTT YSHLLT AQE VSKEQMQIRD ILARPAFYLT ASQQRWEEYL KKGLTNPDAT PEQTRVAVKA IETLNGNWRS PGGAVKFNTV TPSVTGR WF SGNQTWPWDT WKQAFAMAHF NPDIAKENIR AVFSWQIQPG DSVRPQDVGF VPDLIAWNLS PERGGDGGNW NERNTKPS L AAWSVMEVYN VTQDKTWVAE MYPKLVAYHD WWLRNRDHNG NGVPEYGATR DKAHNTESGE MLFTVKKDSL RLSCASSRS IDGINIMRWY RQAPGKQRGM VAVVTGWGST NYVDSVKGRF IISRDSAKDT VYLQMNNLKP EDTAVYSCNA IYRGSEYWGQ GTQVTVSSG ENLYFQGSHH HHHHHHHH

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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
TemperatureMin: 80.0 K / Max: 80.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 585252
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software: (Name: cryoSPARC, RELION)
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8ajb:
Cryo-EM structure of crescentin filaments (stutter mutant, C2 symmetry and large box)

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