EMDB-24098: pKM101-T4SS outer membrane core complex

Basic information

• Entry: Database: EMDB / ID: EMD-24098
• Title: pKM101-T4SS outer membrane core complex
• Map data: pKM101 outer membrane core complex

Sample

• Cell: Type IV secretion machine

• Biological species: Escherichia coli (E. coli)
• Method: subtomogram averaging / cryo EM / Resolution: 37.5 Å
• Authors: Khara P / Christie PJ / Hu B

Funding support

United States, 1 items

Organization	Grant number	Country
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)	5R21AI142378-02	United States

• Citation: Journal: mBio / Year: 2021; Title: Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center.; Authors: Pratick Khara / Liqiang Song / Peter J Christie / Bo Hu / ; Abstract: Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as "minimized" or "expanded" based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical minimized systems mediating Agrobacterium tumefaciens transfer DNA (T-DNA) and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1 to VirB11 and VirD4. We visualized the pKM101-encoded T4SS in its native cellular context by cryo-electron tomography (CryoET). The T4SS is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SS analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three expanded T4SSs previously visualized by CryoET but differs strikingly from the IMC of the purified T4SS, whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SS function supplied evidence that TraB as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers of dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer. Bacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryo-electron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the T4SS differs remarkably from that of a closely related T4SS analyzed by single-particle electron microscopy. Our findings underscore the importance of comparative and analyses of the T4SS nanomachines and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for the development of intervention strategies aimed at suppressing the action of T4SS nanomachines.

History

Deposition	May 24, 2021	-
Header (metadata) release	Dec 15, 2021	-
Map release	Dec 15, 2021	-
Update	Dec 15, 2021	-
Current status	Dec 15, 2021	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_24098.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: pKM101 outer membrane core complex
• Voxel size: X=Y=Z: 5 Å

Density

Contour Level	By AUTHOR: 1.43 / Movie #1: 1.2
Minimum - Maximum	-7.600954 - 6.44264
Average (Standard dev.)	5.6844274e-10 (±0.99999994)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin -40 -40 -40 Dimensions 80 80 80 Spacing 80 80 80
Cell	A=B=C: 400.0 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	5	5	5
M x/y/z	80	80	80
origin x/y/z	0.000	0.000	0.000
length x/y/z	400.000	400.000	400.000
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	-40	-40	-40
NC/NR/NS	80	80	80
D min/max/mean	-7.601	6.443	0.000

Supplemental data

Sample components

Entire : Type IV secretion machine

• Entire: Name: Type IV secretion machine

Components

• Cell: Type IV secretion machine

Supramolecule #1: Type IV secretion machine

• Supramolecule: Name: Type IV secretion machine / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
• Source (natural): Organism: Escherichia coli (E. coli)

Experimental details

Structure determination

• Method: cryo EM
• Processing: subtomogram averaging
• Aggregation state: cell

Sample preparation

• Buffer: pH: 7
• Vitrification: Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

Electron microscopy

• Microscope: FEI POLARA 300
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.7 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: OTHER / Imaging mode: BRIGHT FIELD
• Sample stage: Specimen holder model: OTHER
• Experimental equipment: Model: Tecnai Polara / Image courtesy: FEI Company

Image processing

• Final reconstruction: Applied symmetry - Point group: C₁₄ (14 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 37.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 753
• Extraction: Number tomograms: 1781 / Number images used: 837
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Details: subtomo alignment