EMDB-3460: Cryo-EM structure of Lambda Phage protein GamS bound to RecBCD

Basic information

• Entry: Database: EMDB / ID: 3460
• Title: Cryo-EM structure of Lambda Phage protein GamS bound to RecBCD
• Sample: RecBCD complex bound to inhibitory protein lambda GamS
• Source: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 / ; Enterobacteria phage lambda / virus
• Method: single particle reconstruction, at 3.8 Å resolution
• Authors: Wilkinson M / Chaban Y

Citation

Elife, 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

• Date: Deposition: Nov 8, 2016 / Header (metadata) release: Nov 16, 2016 / Map release: Jan 11, 2017 / Last update: Nov 8, 2016

Map

• File: emd_3460.map.gz (map file in CCP4 format, 23329 KB)
• Voxel size: X=Y=Z: 1.34 Å

Density

Contour Level:	0.035 (by author), 0.035 (movie #1):
Minimum - Maximum	-0.042780045 - 0.16285071
Average (Standard dev.)	-0.0006984152 (0.009656814)

Details

EMDB XML:

Space Group Number	1
Map Geometry	Axis order X Y Z Dimensions 180 180 180 Origin 0 0 0 Limit 179 179 179 Spacing 180 180 180
Cell	A=B=C: 241.20001 Å Alpha=beta=gamma: 90 deg.

CCP4 map header:

mode	Image stored as Reals
A/pix X/Y/Z	1.34	1.34	1.34
M x/y/z	180	180	180
origin x/y/z	0.000	0.000	0.000
length x/y/z	241.200	241.200	241.200
alpha/beta/gamma	90.000	90.000	90.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	180	180	180
D min/max/mean	-0.043	0.163	-0.001

Supplemental data

Sample components

Entire RecBCD complex bound to inhibitory protein lambda GamS

• Entire: Name: RecBCD complex bound to inhibitory protein lambda GamS; Details: RecBCD 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.; Number of components: 7
• Mass: Theoretical: 20 kDa

Component #1: protein, RecBCD complex bound to inhibitory protein lambda GamS

• Protein: Name: RecBCD complex bound to inhibitory protein lambda GamS; Details: RecBCD 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.; Recombinant expression: No
• Mass: Theoretical: 20 kDa

Component #2: protein, RecBCD helicase/nuclease complex

• Protein: Name: RecBCD helicase/nuclease complex / Recombinant expression: No
• Source: Species: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
• Source (engineered): Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 / ; Vector: Multiple / Strain: BL21(DE3)

Component #3: protein, Lambda GamS

• Protein: Name: Lambda GamS / Recombinant expression: No
• Source: Species: Enterobacteria phage lambda / virus
• Source (engineered): Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 / ; Vector: pET22b / Strain: BL21(DE3)

Component #4: protein, RecBCD enzyme subunit RecB

• Protein: Name: RecBCD enzyme subunit RecB / Recombinant expression: No
• Mass: Theoretical: 134.167703 kDa
• Source (engineered): Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 / ; Vector: pETduet-1

Component #5: protein, RecBCD enzyme subunit RecC

• Protein: Name: RecBCD enzyme subunit RecC / Recombinant expression: No
• Mass: Theoretical: 128.974102 kDa
• Source (engineered): Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 / ; Vector: pRSFduet-1

Component #6: protein, RecBCD enzyme subunit RecD

• Protein: Name: RecBCD enzyme subunit RecD / Recombinant expression: No
• Mass: Theoretical: 67.047422 kDa
• Source (engineered): Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 / ; Vector: pCDFduet-1

Component #7: protein, Host-nuclease inhibitor protein gam

• Protein: Name: Host-nuclease inhibitor protein gam; 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.; Recombinant expression: No
• Mass: Theoretical: 11.661924 kDa
• Source (engineered): Expression System: Enterobacteria phage lambda / virus / Vector: pET22b

Experimental details

Sample preparation

• Specimen state: particle
• Sample solution: Specimen conc.: 1.4 mg/ml / pH: 7.5
• Support film: 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.
• Vitrification: Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 90 % / Details: 3 ul sample applied Blot force of -4 for 1 s

Electron microscopy imaging

• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company
• Imaging: Microscope: FEI TITAN KRIOS
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.4 e/Ų / Illumination mode: FLOOD BEAM
• Lens: Magnification: 105000 X (nominal), 37313 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200 - 2400 nm
• Specimen Holder: Model: FEI TITAN KRIOS AUTOGRID HOLDER
• Camera: Detector: GATAN K2 (4k x 4k)

Image acquisition

• Image acquisition: Number of digital images: 334 / Sampling size: 5 microns

Image processing

• Processing: Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 122796; Details: Frames were aligned using motioncorr prior to processing.
• 3D reconstruction: Software: Relion / CTF correction: CTF correction done at start of processing / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF

Atomic model buiding

Modeling #1

Refinement protocol: rigid body / Target criteria: Correlation coefficient
Input PDB model: 5LD2

5ld2

Modeling #2

Refinement protocol: rigid body / Target criteria: Correlation coefficient
Input PDB model: 2UUZ

2uuz

Output model

5mbv