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- EMDB-11986: Cryo-EM structure of exoglucanase Cel48S -

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Basic information

Entry
Database: EMDB / ID: EMD-11986
TitleCryo-EM structure of exoglucanase Cel48S
Map data
Sample
  • Complex: Full-length exoglucanase Cel48S
    • Protein or peptide: exoglucanase Cel48S
Biological speciesHungateiclostridium thermocellum DSM 1313 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsTatli M / Morais S / Tovar-Herrera OE / Bomble YJ / Bayer EA / Medalia O / Mizrahi I
Funding support Switzerland, Israel, Germany, 4 items
OrganizationGrant numberCountry
Swiss National Science FoundationSNSF 31003A_179418/1 Switzerland
European Research Council (ERC)64084 Israel
German Research Foundation (DFG)2476/2 -1 Germany
European Molecular Biology Organization (EMBO)7686
CitationJournal: Elife / Year: 2022
Title: Nanoscale resolution of microbial fiber degradation in action.
Authors: Meltem Tatli / Sarah Moraïs / Omar E Tovar-Herrera / Yannick J Bomble / Edward A Bayer / Ohad Medalia / Itzhak Mizrahi /
Abstract: The lives of microbes unfold at the micron scale, and their molecular machineries operate at the nanoscale. Their study at these resolutions is key toward achieving a better understanding of their ...The lives of microbes unfold at the micron scale, and their molecular machineries operate at the nanoscale. Their study at these resolutions is key toward achieving a better understanding of their ecology. We focus on cellulose degradation of the canonical system to comprehend how microbes build and use their cellulosomal machinery at these nanometer scales. Degradation of cellulose, the most abundant organic polymer on Earth, is instrumental to the global carbon cycle. We reveal that bacterial cells form 'cellulosome capsules' driven by catalytic product-dependent dynamics, which can increase the rate of hydrolysis. Biosynthesis of this energetically costly machinery and cell growth are decoupled at the single-cell level, hinting at a division-of-labor strategy through phenotypic heterogeneity. This novel observation highlights intrapopulation interactions as key to understanding rates of fiber degradation.
History
DepositionNov 26, 2020-
Header (metadata) releaseNov 3, 2021-
Map releaseNov 3, 2021-
UpdateMay 17, 2023-
Current statusMay 17, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0128
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0128
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11986.png

Downloads & links

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Map

FileDownload / File: emd_11986.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.845 Å
Density
Contour LevelBy AUTHOR: 0.0128 / Movie #1: 0.0128
Minimum - Maximum-0.1791954 - 0.21842836
Average (Standard dev.)0.00059506163 (±0.01020033)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 108.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8450.8450.845
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z108.160108.160108.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1790.2180.001

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Supplemental data

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Mask #1

Fileemd_11986_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: #1

Fileemd_11986_additional_1.map
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Additional map: #2

Fileemd_11986_additional_2.map
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Half map: Relion job648 halfmap2

Fileemd_11986_half_map_1.map
AnnotationRelion job648 halfmap2
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Half map: Relion job648 halfmap1

Fileemd_11986_half_map_2.map
AnnotationRelion job648 halfmap1
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Sample components

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Entire : Full-length exoglucanase Cel48S

EntireName: Full-length exoglucanase Cel48S
Components
  • Complex: Full-length exoglucanase Cel48S
    • Protein or peptide: exoglucanase Cel48S

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Supramolecule #1: Full-length exoglucanase Cel48S

SupramoleculeName: Full-length exoglucanase Cel48S / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Hungateiclostridium thermocellum DSM 1313 (bacteria)
Location in cell: Extracellular
Molecular weightTheoretical: 82 KDa

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Macromolecule #1: exoglucanase Cel48S

MacromoleculeName: exoglucanase Cel48S / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: cellulose 1,4-beta-cellobiosidase (reducing end)
Source (natural)Organism: Hungateiclostridium thermocellum DSM 1313 (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGPTKAPTKD GTSYKDLFLE LYGKIKDPKN GYFSPDEGIP YHSIETLIVE APDYGHVTTS EAFSYYVWLE AMYGNLTGNW SGVETAWKVM EDWIIPDSTE QPGMSSYNPN SPATYADEYE DPSYYPSELK FDTVRVGSDP VHNDLVSAYG PNMYLMHWLM DVDNWYGFGT ...String:
MGPTKAPTKD GTSYKDLFLE LYGKIKDPKN GYFSPDEGIP YHSIETLIVE APDYGHVTTS EAFSYYVWLE AMYGNLTGNW SGVETAWKVM EDWIIPDSTE QPGMSSYNPN SPATYADEYE DPSYYPSELK FDTVRVGSDP VHNDLVSAYG PNMYLMHWLM DVDNWYGFGT GTRATFINTF QRGEQESTWE TIPHPSIEEF KYGGPNGFLD LFTKDRSYAK Q WRYTNAPD AEGRAIQAVY WANKWAKEQG KGSAVASVVS KAAKMGDFLR NDMFDKYFMK IGAQDKTPAT GYDSAHYLMA WYTAWGGGIG ASWAWKIGCS HAHFGYQNPF QGWVSATQSD FAPKSSNGKR DWTTSYKRQL EFYQWLQSAE GGIAGGATNS WNGRYEKYPA GTSTFYGMAY VPHPVYADPG SNQWFGFQAW SMQRVMEYYL ETGDSSVKNL IKKWVDWVMS EIKLYDDGTF AIPSDLEWSG QPDTWTGTYT GNPNLHVRVT SYGTDLGVAG SLANALATYA AATERWEGKL DTKARDMAAE LVNRAWYNFY CSEGKGVVTE EARADYKRFF EQEVYVPAGW SGTMPNGDKI QPGIKFIDIR TKYRQDPYYD IVYQAYLRGE APVLNYHRFW HEVDLAVAMG VLATYFPDMT YKVPGTPSTK LYGDVNDDGK VNSTDAVALK RYVLRSGISI NTDNADLNED GRVNSTDLGI LKRYILKEID TLPYKNHHHH HH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.35 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMTris/HClTris(hydroxymethyl)aminomethane hydrochloride
2.7 mMKClPotassium chloride
137.0 mMNaClSodium chlorideSodium chloride
2.5 mMCaCl2Calcium chloride
2.0 mM2-MercaptoethanolBeta-Mercaptoethanol2-Mercaptoethanol
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 4-5 s blotting time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal magnification: 58180
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-60 / Number real images: 2000 / Average exposure time: 12.0 sec. / Average electron dose: 67.0 e/Å2
Details: Images were collected in movie-mode at 5 frames per second
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1331906
Startup modelType of model: OTHER
Details: Stochastic Gradient Descent (SGD) based initial model building of RELION 3.0
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 55245
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 111 / Target criteria: 0.87

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