[English] 日本語
Yorodumi
- PDB-5mbv: Cryo-EM structure of Lambda Phage protein GamS bound to RecBCD -

+
Open data


ID or keywords:

Loading...

no data

-
Basic information

Entry
Database: PDB / ID: 5mbv
TitleCryo-EM structure of Lambda Phage protein GamS bound to RecBCD
DescriptorHost-nuclease inhibitor protein gam
(RecBCD enzyme subunit ...) x 3
KeywordsDNA BINDING PROTEIN / Inhibitor / Complex / DNA repair / Helicase/Nuclease / DNA binding protein
Specimen sourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Enterobacteria phage lambda / virus
MethodElectron microscopy (3.8 Å resolution / Particle / Single particle)
AuthorsWilkinson, M. / Chaban, Y. / Wigley, D.B.
CitationElife, 2016, 5

Elife, 2016, 5 StrPapers
Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones.
Martin Wilkinson / Lucy Troman / Wan Ak Wan Nur Ismah / Yuriy Chaban / Matthew B Avison / Mark S Dillingham / Dale B Wigley

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 8, 2016 / Release: Jan 11, 2017

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
3D viewer


View / / Stereo:
Center
Zoom
Scale
Slabnear <=> far

fix: /
Orientation
Orientation Rotation
Misc. /
Show/hide

Downloads & links

-
Assembly

Deposited unit
B: RecBCD enzyme subunit RecB
C: RecBCD enzyme subunit RecC
D: RecBCD enzyme subunit RecD
A: Host-nuclease inhibitor protein gam
E: Host-nuclease inhibitor protein gam


Theoretical massNumber of molelcules
Total (without water)353,5135
Polyers353,5135
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)25710
ΔGint (kcal/M)-97
Surface area (Å2)129280
MethodPISA

-
Components

#1: Polypeptide(L)RecBCD enzyme subunit RecB / Exodeoxyribonuclease V 135 kDa polypeptide / Exodeoxyribonuclease V beta chain / Exonuclease V subunit RecB / ExoV subunit RecB / Helicase/nuclease RecBCD subunit RecB


Mass: 134167.703 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
References: UniProt: P08394, EC: 3.1.11.5

Cellular component

Molecular function

Biological process

#2: Polypeptide(L)RecBCD enzyme subunit RecC / Exodeoxyribonuclease V 125 kDa polypeptide / Exodeoxyribonuclease V gamma chain / Exonuclease V subunit RecC / ExoV subunit RecC


Mass: 128974.102 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
References: UniProt: P07648, EC: 3.1.11.5

Cellular component

Molecular function

Biological process

#3: Polypeptide(L)RecBCD enzyme subunit RecD / Exodeoxyribonuclease V 67 kDa polypeptide / Exodeoxyribonuclease V alpha chain / Exonuclease V subunit RecD / ExoV subunit RecD / Helicase/nuclease RecBCD subunit RecD


Mass: 67047.422 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
References: UniProt: P04993, EC: 3.1.11.5

Cellular component

Molecular function

Biological process

#4: Polypeptide(L)Host-nuclease inhibitor protein gam


Mass: 11661.924 Da / Num. of mol.: 2
Details: GamL is cleaved at position Y44 when expressed in E.coli into GamS (see paper - pubmed id 17544443). We had the GamS gene synthesised to start at M41 for this project. It purifies as a dimer.
Mutation: L79I same as crystal structure / Source: (gene. exp.) Enterobacteria phage lambda / virus / References: UniProt: P03702

Molecular function

Biological process

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

-
Sample preparation

Component

Type: COMPLEX

IDNameDetailsEntity IDParent IDSource
1RecBCD complex bound to inhibitory protein lambda GamSRecBCD and GamS were expressed and purified separately. They were mixed with an excess of GamS then passed through a size exclusion column to separate out free GamS.1, 2, 3, 4, 50MULTIPLE SOURCES
2RecBCD helicase/nuclease complex1, 2, 31RECOMBINANT
3Lambda GamS4, 51RECOMBINANT
Molecular weight
IDValueUnitsEntity assembly IDExperimental flag
10.35MEGADALTONS1NO
20.33MEGADALTONS1NO
30.02MEGADALTONS1NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
22562Escherichia coli
3310710Enterobacteria phage lambda
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismPlasmidStrain
22562Escherichia coliMultipleBL21(DE3)
33562Escherichia colipET22bBL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.Conc. unitsNameBuffer ID
120mMTris-HCl1
250mMSodium chloride1
30.5mMTCEP-HCl1
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were thinned by glow discharge in 30s steps with 1 minute wait in between treatments. Then left for 1-2 weeks prior to overnight treatment with 1 mM Ampiphol A8-35 to render surface hydrophilic. Grids were washed with 5 drops of water prior to use.
Grid mesh size: 400 / Grid type: EMS C-Flat 1/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Details: 3 ul sample applied Blot force of -4 for 1 s / Chamber temperature: 277 kelvins

-
Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 / Calibrated magnification: 37313 / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Calibrated defocus min: 600 nm / Calibrated defocus max: 2900 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.4 sec. / Electron dose: 1.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 334
Image scansSampling size: 5 microns / Movie frames/image: 25 / Used frames/image: 1-25

-
Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetailsImage processing IDImaging IDFitting ID
1GautomatchPARTICLE SELECTIONTemplate-free using circles with diameter of 140 Angstroms1
2EPUIMAGE ACQUISITIONFocused every 5 um1
4Gctf0.5CTF CORRECTION1
7CootMODEL FITTING1
9Relion1.4INITIAL EULER ASSIGNMENT1
10Relion1.4FINAL EULER ASSIGNMENT1
11Relion1.4CLASSIFICATION1
12Relion1.4RECONSTRUCTION1
13RefmacMODEL REFINEMENTJelly body refinement1
14PhenixMODEL REFINEMENTReal-space refinement1
Image processingDetails: Frames were aligned using motioncorr prior to processing.
CTF correctionDetails: CTF correction done at start of processing / Type: PHASE FLIPPING ONLY
Particle selectionNumber of particles selected: 134124
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 122796 / Symmetry type: POINT
Atomic model building
IDRef protocolTarget criteria
1RIGID BODY FITCorrelation coefficient
2RIGID BODY FITCorrelation coefficient
Atomic model building
IDPDB-ID 3D fitting ID
15LD21
22UUZ2
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00124635
ELECTRON MICROSCOPYf_angle_d0.42733407
ELECTRON MICROSCOPYf_dihedral_angle_d15.51414917
ELECTRON MICROSCOPYf_chiral_restr0.0363657
ELECTRON MICROSCOPYf_plane_restr0.0034402

+
About Yorodumi

-
News

-
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. See below links for details.
  • 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

-
Jun 16, 2017. Omokage search with filter

Omokage search with filter

  • Result of Omokage search can be filtered by keywords and the database types

Related info.: Omokage search

+
Sep 15, 2016. EM Navigator & Yorodumi renewed

EM Navigator & Yorodumi renewed

  • New versions of EM Navigator and Yorodumi started

Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator (legacy version) / Yorodumi (legacy version)

+
Aug 31, 2016. New EM Navigator & Yorodumi

New EM Navigator & Yorodumi

  • In 15th Sep 2016, the development versions of EM Navigator and Yorodumi will replace the official versions.
  • Current version will continue as 'legacy version' for some time.

Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator / Yorodumi / EM Navigator (legacy version) / Yorodumi (legacy version)

+
Apr 13, 2016. Omokage search got faster

Omokage search got faster

  • The computation time became ~1/2 compared to the previous version by re-optimization of data accession
  • Enjoy "shape similarity" of biomolecules, more!

Related info.: Omokage search

Read more

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • All the functionalities will be ported from the levgacy version.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.

Related info.: Yorodumi (legacy version) / EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Yorodumi Papers / Jmol/JSmol / Changes in new EM Navigator and Yorodumi

Read more