[English] 日本語
Yorodumi- EMDB-15399: Cryo-EM map of crescentin filament in complex with a megabody (st... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-15399 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM map of crescentin filament in complex with a megabody (stutter mutant, C2 symmetry, large box) | |||||||||
Map data | ||||||||||
Sample |
| |||||||||
Keywords | cytoskeleton / cell shape / intermediate filaments / coiled coil / assembly / STRUCTURAL PROTEIN | |||||||||
Biological species | Caulobacter vibrioides (bacteria) / Camelidae (mammal) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.1 Å | |||||||||
Authors | Liu Y / Lowe J | |||||||||
Funding support | United Kingdom, 1 items
| |||||||||
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 |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_15399.map.gz | 735.8 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-15399-v30.xml emd-15399.xml | 18.5 KB 18.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_15399_fsc.xml | 20.9 KB | Display | FSC data file |
Images | emd_15399.png | 31.4 KB | ||
Masks | emd_15399_msk_1.map | 824 MB | Mask map | |
Filedesc metadata | emd-15399.cif.gz | 5.8 KB | ||
Others | emd_15399_half_map_1.map.gz emd_15399_half_map_2.map.gz | 765.8 MB 765.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-15399 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-15399 | HTTPS FTP |
-Validation report
Summary document | emd_15399_validation.pdf.gz | 655.1 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_15399_full_validation.pdf.gz | 654.7 KB | Display | |
Data in XML | emd_15399_validation.xml.gz | 28 KB | Display | |
Data in CIF | emd_15399_validation.cif.gz | 37.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15399 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15399 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|
-Map
File | Download / File: emd_15399.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.59 Å | ||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Mask #1
File | emd_15399_msk_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: #1
File | emd_15399_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: #2
File | emd_15399_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
-Entire : Crescentin filament in complex with megabody MB13
Entire | Name: Crescentin filament in complex with megabody MB13 |
---|---|
Components |
|
-Supramolecule #1: Crescentin filament in complex with megabody MB13
Supramolecule | Name: Crescentin filament in complex with megabody MB13 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
---|
-Supramolecule #2: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)
Supramolecule | Name: Caulobacter crescentus strain NA1000 crescentin (stutter mutant) type: complex / ID: 2 / Parent: 1 |
---|---|
Source (natural) | Organism: Caulobacter vibrioides (bacteria) |
-Supramolecule #3: Crescentin-specific megabody MB13
Supramolecule | Name: Crescentin-specific megabody MB13 / type: complex / ID: 3 / Parent: 1 |
---|---|
Source (natural) | Organism: Camelidae (mammal) |
-Macromolecule #1: Caulobacter crescentus strain NA1000 crescentin (stutter mutant)
Macromolecule | Name: Caulobacter crescentus strain NA1000 crescentin (stutter mutant) type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Caulobacter vibrioides (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #2: Crescentin-specific megabody MB13
Macromolecule | Name: Crescentin-specific megabody MB13 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Camelidae (mammal) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 2 mg/mL |
---|---|
Buffer | pH: 6.5 / Details: PIPES 25mM pH 6.5, 0.05% CHAPS |
Grid | Model: UltrAuFoil R2/2 / Material: GOLD / Mesh: 300 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Temperature | Min: 80.0 K / Max: 80.0 K |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 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 stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |