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- PDB-8odn: RcpA-TadD with C13 symmetry from the Pseudomonas aeruginosa Tight... -

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Basic information

Entry
Database: PDB / ID: 8odn
TitleRcpA-TadD with C13 symmetry from the Pseudomonas aeruginosa Tight Adherence Secretion System
Components
  • RcpA
  • TPR repeat-containing protein PA4299
KeywordsMEMBRANE PROTEIN / Secretin / Pilotin / RcpA / TadD / TAD
Function / homology
Function and homology information


type II protein secretion system complex / protein secretion / plasma membrane
Similarity search - Function
Uncharacterised conserved protein UCP029658, TPR / Pilus formation protein, N-terminal / Pilus formation protein N terminal region / GspD/PilQ family / Type II/III secretion system / Bacterial type II and III secretion system protein / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeat / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
RcpA / TPR repeat-containing protein PA4299
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsTassinari, M. / Low, H.H.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust215553/Z/19/Z United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Assembly mechanism of a Tad secretion system secretin-pilotin complex.
Authors: Matteo Tassinari / Marta Rudzite / Alain Filloux / Harry H Low /
Abstract: The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly ...The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly unknown. This includes characterisation of the outer membrane secretin through which the pilus is channelled and recruitment of its pilotin. Here we investigate RcpA and TadD lipoprotein from Pseudomonas aeruginosa. Light microscopy reveals RcpA colocalising with TadD in P. aeruginosa and when heterologously expressed in Escherichia coli. We use cryogenic electron microscopy to determine how RcpA and TadD assemble a secretin channel with C13 and C14 symmetries. Despite low sequence homology, we show that TadD shares a similar fold to the type 4 pilus system pilotin PilF. We establish that the C-terminal four residues of RcpA bind TadD - an interaction essential for secretin formation. The binding mechanism between RcpA and TadD appears distinct from known secretin-pilotin pairings in other secretion systems.
History
DepositionMar 9, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RcpA
B: TPR repeat-containing protein PA4299
C: RcpA
D: TPR repeat-containing protein PA4299
E: RcpA
F: TPR repeat-containing protein PA4299
G: RcpA
H: TPR repeat-containing protein PA4299
I: RcpA
J: TPR repeat-containing protein PA4299
K: RcpA
L: TPR repeat-containing protein PA4299
M: RcpA
N: TPR repeat-containing protein PA4299
O: RcpA
P: TPR repeat-containing protein PA4299
Q: RcpA
R: TPR repeat-containing protein PA4299
S: RcpA
T: TPR repeat-containing protein PA4299
V: RcpA
W: TPR repeat-containing protein PA4299
X: RcpA
Y: TPR repeat-containing protein PA4299
Z: RcpA
a: TPR repeat-containing protein PA4299


Theoretical massNumber of molelcules
Total (without water)952,47226
Polymers952,47226
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, 2D class averages end views
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "O"
d_2ens_1chain "C"
d_3ens_1chain "E"
d_4ens_1chain "G"
d_5ens_1chain "I"
d_6ens_1chain "K"
d_7ens_1chain "M"
d_8ens_1chain "A"
d_9ens_1chain "Q"
d_10ens_1chain "S"
d_11ens_1chain "V"
d_12ens_1chain "X"
d_13ens_1chain "Z"
d_1ens_2chain "W"
d_2ens_2chain "D"
d_3ens_2chain "F"
d_4ens_2chain "H"
d_5ens_2chain "J"
d_6ens_2chain "L"
d_7ens_2chain "N"
d_8ens_2chain "P"
d_9ens_2chain "R"
d_10ens_2chain "T"
d_11ens_2chain "B"
d_12ens_2chain "Y"
d_13ens_2chain "a"

NCS domain segments:

Component-ID: 1

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_1ens_1PROPROASPASPOO131 - 416131 - 416
d_2ens_1PROPROASPASPCC131 - 416131 - 416
d_3ens_1PROPROASPASPEE131 - 416131 - 416
d_4ens_1PROPROASPASPGG131 - 416131 - 416
d_5ens_1PROPROASPASPII131 - 416131 - 416
d_6ens_1PROPROASPASPKK131 - 416131 - 416
d_7ens_1PROPROASPASPMM131 - 416131 - 416
d_8ens_1PROPROASPASPAA131 - 416131 - 416
d_9ens_1PROPROASPASPQQ131 - 416131 - 416
d_10ens_1PROPROASPASPSS131 - 416131 - 416
d_11ens_1PROPROASPASPVU131 - 416131 - 416
d_12ens_1PROPROASPASPXW131 - 416131 - 416
d_13ens_1PROPROASPASPZY131 - 416131 - 416
d_1ens_2GLUGLUGLYGLYWV28 - 21228 - 212
d_2ens_2GLUGLUGLYGLYDD28 - 21228 - 212
d_3ens_2GLUGLUGLYGLYFF28 - 21228 - 212
d_4ens_2GLUGLUGLYGLYHH28 - 21228 - 212
d_5ens_2GLUGLUGLYGLYJJ28 - 21228 - 212
d_6ens_2GLUGLUGLYGLYLL28 - 21228 - 212
d_7ens_2GLUGLUGLYGLYNN28 - 21228 - 212
d_8ens_2GLUGLUGLYGLYPP28 - 21228 - 212
d_9ens_2GLUGLUGLYGLYRR28 - 21228 - 212
d_10ens_2GLUGLUGLYGLYTT28 - 21228 - 212
d_11ens_2GLUGLUGLYGLYBB28 - 21228 - 212
d_12ens_2GLUGLUGLYGLYYX28 - 21228 - 212
d_13ens_2GLUGLUGLYGLYaZ28 - 21228 - 212

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(-0.970944246084, 0.239305810333, 1.16685344114E-5), (-0.239305810085, -0.970944246022, 1.9404385691E-5), (1.59730785881E-5, 1.60482285557E-5, 0.999999999744)342.866008392, 437.618395872, -0.00612467967943
2given(-0.748480975341, 0.663156263299, 7.63643815484E-7), (-0.663156263292, -0.748480975337, 3.84240255506E-6), (3.11968618833E-6, 2.3695500329E-6, 0.999999999992)214.892071207, 477.51952035, -0.00107219854468
3given(-0.3545948946, 0.935020032258, -4.60616000143E-7), (-0.935020032256, -0.354594894598, 2.41105588286E-6), (2.09105346734E-6, 1.28563329397E-6, 0.999999999997)83.0779193696, 453.353936521, -0.000669600157835
4given(0.120555860366, 0.992706545023, 3.38700925536E-7), (-0.992706545023, 0.120555860366, 2.17056188193E-7), (1.74640717172E-7, -3.623980211E-7, 1)-22.4278219918, 370.698061874, 2.02498713122E-5
5given(-0.748517045211, -0.663115550219, 9.29554610509E-6), (0.663115550249, -0.748517045242, 2.69991072832E-7), (6.77883942567E-6, 6.36611409041E-6, 0.999999999957)477.507888435, 214.907088145, -0.00252717987442
6given(0.568076867226, 0.822975499548, 8.14966064627E-6), (-0.822975499569, 0.568076867253, -1.21684822415E-6), (-5.63106996425E-6, -6.01570771461E-6, 0.999999999966)-77.4290899162, 248.482519464, 0.00221726144872
7given(0.885457824738, 0.464719744094, 7.73916283281E-6), (-0.464719744128, 0.885457824748, 3.2800033797E-6), (-5.32841995607E-6, -6.50084642915E-6, 0.999999999965)-69.3340231331, 114.700464579, 0.00222918443384
8given(-0.970934301493, -0.239346154929, 1.74826814713E-5), (0.239346155135, -0.970934301539, 1.08030526454E-5), (1.43888660112E-5, 1.46734669659E-5, 0.999999999789)437.641736985, 342.838494207, -0.00555240784763
9given(0.885452256606, -0.464730353291, -1.14753429387E-6), (0.464730353288, 0.885452256606, -2.13864985207E-6), (2.00998233136E-6, 1.3603783198E-6, 0.999999999997)114.69627362, -69.3374333862, -0.000663841678374
10given(0.568085938799, -0.822969237663, 1.82775026575E-7), (0.822969237657, 0.568085938794, -3.98702251532E-6), (3.17736495741E-6, 2.41538965291E-6, 0.999999999992)248.474656053, -77.4384074034, -0.0010923511534
11given(0.120508079459, -0.99271234635, 1.3745302545E-5), (0.992712346316, 0.120508079208, -1.78040412278E-5), (1.60178715339E-5, 1.57906623553E-5, 0.999999999747)370.682819787, -22.4232111386, -0.00609279397452
12given(-0.354651949844, -0.934998392572, 1.89582745546E-5), (0.934998392646, -0.354651950049, -8.67959672718E-6), (1.48389980284E-5, 1.46477203327E-5, 0.999999999783)453.336342652, 83.0905547821, -0.00564749834331
13given(0.120506525811, 0.992712534979, 1.15516260209E-5), (-0.992712535011, 0.1205065257, 9.83193919186E-6), (8.36824296095E-6, -1.26522567847E-5, 0.999999999885)-22.4275560265, 370.681931569, 0.00116469352722
14given(0.568086121713, 0.822969111399, 5.82355642727E-7), (-0.822969111398, 0.56808612171, 2.36165957383E-6), (1.61274472237E-6, -1.82088673392E-6, 0.999999999997)-77.4399602399, 248.473697974, 0.000117190154015
15given(0.885451964362, 0.464730910105, 9.30397583042E-7), (-0.464730910106, 0.88545196436, 1.96903523571E-6), (9.12491695814E-8, -2.17587063289E-6, 0.999999999998)-69.3382742773, 114.695665399, 0.000366723707828
16given(-0.354652198985, 0.934998298173, 1.30255600242E-5), (-0.934998298229, -0.354652199057, 3.68097067353E-6), (8.06124482189E-6, -1.08734121123E-5, 0.999999999908)83.0856432777, 453.336953687, 0.000893576418861
17given(-0.748517274301, 0.663115291664, 5.90204572642E-6), (-0.663115291682, -0.748517274307, -1.62139201853E-6), (3.34261333871E-6, -5.12737670768E-6, 0.999999999981)214.90467535, 477.508589056, 0.000477998472775
18given(0.885457794802, -0.46471980117, 5.04670326517E-6), (0.464719801188, 0.8854577948, -3.35786838779E-6), (-2.90817481466E-6, 5.31855367594E-6, 0.999999999982)114.700242335, -69.3325408737, -0.000564844820815
19given(0.56807704478, -0.822975377007, 5.85032843325E-6), (0.82297537702, 0.568077044791, 2.96098820132E-7), (-3.56711932554E-6, 4.64646930534E-6, 0.999999999983)248.481959737, -77.427386564, -0.000366728204028
20given(0.120556048262, -0.992706522205, 5.0163729241E-9), (0.992706522205, 0.120556048262, 3.9154601764E-7), (-3.89295039551E-7, -4.22234544797E-8, 1)370.698208675, -22.425747755, 5.54031167326E-5
21given(-0.354595282619, -0.935019885106, -9.00419533828E-7), (0.935019885106, -0.354595282618, -1.23878360386E-6), (8.39002783879E-7, -1.28117699118E-6, 0.999999999999)453.353670485, 83.0807125222, 0.000118969490217
22given(-0.74848075641, -0.663156510398, -3.71068032279E-7), (0.663156510397, -0.748480756408, -2.30512308377E-6), (1.25092009879E-6, -1.97141645076E-6, 0.999999999997)477.518545641, 214.895050618, 0.000188016175969
23given(-0.970943987082, -0.239306861403, 5.8393191246E-6), (0.239306861306, -0.970943987025, -1.38524270364E-5), (8.98463262925E-6, -1.20525416056E-5, 0.999999999887)437.616553579, 342.870821952, 0.00098440265748
24given(-0.970934465907, 0.239345488361, 1.07636474226E-5), (-0.239345488457, -0.970934465903, -8.79683726712E-6), (8.3453129497E-6, -1.11173829436E-5, 0.999999999903)342.835040503, 437.645677364, 0.00090088451455

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Components

#1: Protein
RcpA


Mass: 45507.402 Da / Num. of mol.: 13
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Gene: rcpA / Production host: Escherichia coli (E. coli) / References: UniProt: Q9HW96
#2: Protein
TPR repeat-containing protein PA4299


Mass: 27759.648 Da / Num. of mol.: 13
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Gene: PA4299 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9HWA1

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RcpA-TadD with C13 symmetry / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.9 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: 50 mM Hepes pH 8, 100 mM NaCl, 1 mM EDTA, 0.06% bDDM
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMhepesC8H18N2O4S1
2100 mMsodium chlorideNaCl1
31 mMEDTAC10H16N2O81
40.06 %bDDMC24H46O111
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was coated with graphene oxide prior to use
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
SymmetryPoint symmetry: C13 (13 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119534 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 93.84 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002743225
ELECTRON MICROSCOPYf_angle_d0.612158292
ELECTRON MICROSCOPYf_chiral_restr0.07976539
ELECTRON MICROSCOPYf_plane_restr0.00447722
ELECTRON MICROSCOPYf_dihedral_angle_d12.943316276
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2OOELECTRON MICROSCOPYNCS constraints3.32744969378E-11
ens_1d_3OOELECTRON MICROSCOPYNCS constraints7.58815530537E-10
ens_1d_4OOELECTRON MICROSCOPYNCS constraints1.8008295884E-10
ens_1d_5OOELECTRON MICROSCOPYNCS constraints1.69938496084E-12
ens_1d_6OOELECTRON MICROSCOPYNCS constraints1.23017146914E-12
ens_1d_7OOELECTRON MICROSCOPYNCS constraints9.0695439632E-12
ens_1d_8OOELECTRON MICROSCOPYNCS constraints2.35791778316E-10
ens_1d_9OOELECTRON MICROSCOPYNCS constraints4.97469138198E-12
ens_1d_10OOELECTRON MICROSCOPYNCS constraints3.10950589971E-12
ens_1d_11OOELECTRON MICROSCOPYNCS constraints2.28167453566E-10
ens_1d_12OOELECTRON MICROSCOPYNCS constraints6.37176678006E-13
ens_1d_13OOELECTRON MICROSCOPYNCS constraints8.86019635488E-11
ens_2d_2VWELECTRON MICROSCOPYNCS constraints4.12004130966E-13
ens_2d_3VWELECTRON MICROSCOPYNCS constraints8.13728542701E-11
ens_2d_4VWELECTRON MICROSCOPYNCS constraints1.00349205616E-10
ens_2d_5VWELECTRON MICROSCOPYNCS constraints2.1200794907E-10
ens_2d_6VWELECTRON MICROSCOPYNCS constraints3.51827909536E-13
ens_2d_7VWELECTRON MICROSCOPYNCS constraints5.93781992476E-11
ens_2d_8VWELECTRON MICROSCOPYNCS constraints1.25653890797E-12
ens_2d_9VWELECTRON MICROSCOPYNCS constraints9.94989631398E-12
ens_2d_10VWELECTRON MICROSCOPYNCS constraints3.70058489805E-13
ens_2d_11VWELECTRON MICROSCOPYNCS constraints2.76029963028E-10
ens_2d_12VWELECTRON MICROSCOPYNCS constraints1.87693856082E-12
ens_2d_13VWELECTRON MICROSCOPYNCS constraints1.04397196385E-12

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