[English] 日本語
Yorodumi
- EMDB-26593: CryoEM structure of full-length dimeric ClbP -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-26593
TitleCryoEM structure of full-length dimeric ClbP
Map dataFinal map for ClbP
Sample
  • Complex: ClbP
    • Protein or peptide: Beta-lactamase
Function / homology
Function and homology information


antibiotic catabolic process / beta-lactamase activity / beta-lactamase / outer membrane-bounded periplasmic space / response to antibiotic / membrane
Similarity search - Function
Beta-lactamase, class-C active site / Beta-lactamase class-C active site. / Beta-lactamase-related / Beta-lactamase / Beta-lactamase/transpeptidase-like
Similarity search - Domain/homology
Biological speciesEscherichia coli CFT073 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsVelilla JA / Walsh Jr RM / Gaudet R
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM120996 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA208834 United States
CitationJournal: Nat Chem Biol / Year: 2023
Title: Structural basis of colibactin activation by the ClbP peptidase.
Authors: José A Velilla / Matthew R Volpe / Grace E Kenney / Richard M Walsh / Emily P Balskus / Rachelle Gaudet /
Abstract: Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the ...Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.
History
DepositionApr 4, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateFeb 15, 2023-
Current statusFeb 15, 2023Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_26593.png

Downloads & links

-
Map

FileDownload / File: emd_26593.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map for ClbP
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.245
Minimum - Maximum-3.454813 - 4.3565187
Average (Standard dev.)0.0058111185 (±0.10340754)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: Half map A for ClbP

Fileemd_26593_half_map_1.map
AnnotationHalf map A for ClbP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: Half map B for ClbP

Fileemd_26593_half_map_2.map
AnnotationHalf map B for ClbP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : ClbP

EntireName: ClbP
Components
  • Complex: ClbP
    • Protein or peptide: Beta-lactamase

-
Supramolecule #1: ClbP

SupramoleculeName: ClbP / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all / Details: Full-length ClbP
Source (natural)Organism: Escherichia coli CFT073 (bacteria)
Molecular weightTheoretical: 110 KDa

-
Macromolecule #1: Beta-lactamase

MacromoleculeName: Beta-lactamase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: beta-lactamase
Source (natural)Organism: Escherichia coli CFT073 (bacteria)
Molecular weightTheoretical: 53.523309 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: QEHEPIGAQD ERLSTLIHQR MQEAKVPALS VSVTIKGVRQ RFVYGVADVA SQKANTLDTV YELGSMSKAF TGLVVQILIQ EGRLRQGDD IITYLPEMRL NYQGKPASLT VADFLYHTSG LPFSTLARLE NPMPGSAVAQ QLRNENLLFA PGAKFSYASA N YDVLGAVI ...String:
QEHEPIGAQD ERLSTLIHQR MQEAKVPALS VSVTIKGVRQ RFVYGVADVA SQKANTLDTV YELGSMSKAF TGLVVQILIQ EGRLRQGDD IITYLPEMRL NYQGKPASLT VADFLYHTSG LPFSTLARLE NPMPGSAVAQ QLRNENLLFA PGAKFSYASA N YDVLGAVI ENVTGKTFTE VIAERLTQPL GMSATVAVKG DEIIVNKASG YKLGFGKPVL FHAPLARNHV PAAYIHSTLP DM EIWIDAW LHRKALPATL REAMSNSWRG NSDVPLAADN RILYASGWFI DQNQGPYISH GGQNPNFSSC IALRPDQQIG IVA LANMNS NLILQLCADI DNYLRIGKYA DGAGDAITAT DTLFVYLTLL LCFWGAVVVV RGAFRVYRAT AHGPGKQQRL RLRV RDYII ALAVPGLVAA MLYVAPGILS PGLDWRFILV WGPSSVLAIP FGIILLAFVL TLNHQIKRIL LHNKEWDDEH HHHHH HHHH

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration3.5 mg/mL
BufferpH: 7.3
Component:
ConcentrationNameFormula
10.0 mMHEPES
200.0 mMsodium chlorideNaClSodium chloride
0.06 %GDN
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa / Details: 30 s glow discharge at 15mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: Three uL of sample were deposited onto 400 mesh Quantifoil Cu 1.2/1.3 grids that had been glow discharged in a PELCO easiGLOW (Ted Pella) at 0.39 mBar, 15 mA for 30 s. Samples were vitrified ...Details: Three uL of sample were deposited onto 400 mesh Quantifoil Cu 1.2/1.3 grids that had been glow discharged in a PELCO easiGLOW (Ted Pella) at 0.39 mBar, 15 mA for 30 s. Samples were vitrified in 100% liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific), with a wait time of 30 s, blot time of 5 s and a blot force of 16 at 100% humidity..
DetailsProtein was purified by Ni affinity chromatography followed by SEC on an S200 10/300 column equilibrated with 10 mM HEPES pH 7.3, 200 mM NaCl, 0.06% GDN. Sample used for preparing grids came from the peak fraction and was not concentrated.

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 60606 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3888 / Average exposure time: 2.5 sec. / Average electron dose: 76.191 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Particle selectionNumber selected: 562462
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 109906
FSC plot (resolution estimation)

-
Atomic model buiding 1

Initial modelPDB ID:

7mdf


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7ul6:
CryoEM structure of full-length dimeric ClbP

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more