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- PDB-5lfp: Crystal Structure of the Bacterial Proteasome Activator Bpa of My... -

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Basic information

Entry
Database: PDB / ID: 5lfp
TitleCrystal Structure of the Bacterial Proteasome Activator Bpa of Mycobacterium tuberculosis (space group P6322, SeMet)
ComponentsBacterial proteasome activator
KeywordsCHAPERONE / Dodecamer / four-helix bundle
Function / homologyBacterial proteasome activator / Bacterial proteasome activator / proteasome accessory complex / positive regulation of proteasomal protein catabolic process / proteasome binding / peptidoglycan-based cell wall / protein homooligomerization / plasma membrane / Bacterial proteasome activator
Function and homology information
Biological speciesMycobacterium tuberculosis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3.303 Å
AuthorsBolten, M. / Delley, C.L. / Leibundgut, M. / Boehringer, D. / Ban, N. / Weber-Ban, E.
CitationJournal: Structure / Year: 2016
Title: Structural Analysis of the Bacterial Proteasome Activator Bpa in Complex with the 20S Proteasome.
Authors: Marcel Bolten / Cyrille L Delley / Marc Leibundgut / Daniel Boehringer / Nenad Ban / Eilika Weber-Ban /
Abstract: Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial ...Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial proteasome activator) was shown to support an alternate proteasomal degradation pathway. Here, we present the cryo-electron microscopy (cryo-EM) structure of Bpa in complex with the 20S core particle (CP). For docking into the cryo-EM density, we solved the X-ray structure of Bpa, showing that it forms tight four-helix bundles arranged into a 12-membered ring with a 40 Å wide central pore and the C-terminal helix of each protomer protruding from the ring. The Bpa model was fitted into the cryo-EM map of the Bpa-CP complex, revealing its architecture and striking symmetry mismatch. The Bpa-CP interface was resolved to 3.5 Å, showing the interactions between the C-terminal GQYL motif of Bpa and the proteasome α-rings. This docking mode is related to the one observed for eukaryotic activators with features specific to the bacterial complex.
History
DepositionJul 4, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 23, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2016Group: Database references

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bacterial proteasome activator
B: Bacterial proteasome activator
C: Bacterial proteasome activator
D: Bacterial proteasome activator


Theoretical massNumber of molelcules
Total (without water)60,0654
Polymers60,0654
Non-polymers00
Water00
1
A: Bacterial proteasome activator
B: Bacterial proteasome activator
C: Bacterial proteasome activator
D: Bacterial proteasome activator

A: Bacterial proteasome activator
B: Bacterial proteasome activator
C: Bacterial proteasome activator
D: Bacterial proteasome activator

A: Bacterial proteasome activator
B: Bacterial proteasome activator
C: Bacterial proteasome activator
D: Bacterial proteasome activator


Theoretical massNumber of molelcules
Total (without water)180,19612
Polymers180,19612
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-y+1,x-y+1,z1
crystal symmetry operation3_565-x+y,-x+1,z1
Buried area25840 Å2
ΔGint-143 kcal/mol
Surface area57570 Å2
MethodPISA
Unit cell
Length a, b, c (Å)100.857, 100.857, 207.422
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resseq 39:105 or resseq 107:138 or (resid...
21(chain B and (resseq 39:105 or resseq 107:138 or (resid...
31(chain C and (resseq 39:105 or resseq 107:138 or (resid...
41(chain D and (resseq 39:105 or resseq 107:138 or (resid...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resseq 39:105 or resseq 107:138 or (resid...A39 - 105
121(chain A and (resseq 39:105 or resseq 107:138 or (resid...A107 - 138
131(chain A and (resseq 39:105 or resseq 107:138 or (resid...A139
141(chain A and (resseq 39:105 or resseq 107:138 or (resid...A37 - 144
151(chain A and (resseq 39:105 or resseq 107:138 or (resid...A37 - 144
161(chain A and (resseq 39:105 or resseq 107:138 or (resid...A37 - 144
211(chain B and (resseq 39:105 or resseq 107:138 or (resid...B39 - 105
221(chain B and (resseq 39:105 or resseq 107:138 or (resid...B107 - 138
231(chain B and (resseq 39:105 or resseq 107:138 or (resid...B139
241(chain B and (resseq 39:105 or resseq 107:138 or (resid...B37 - 144
251(chain B and (resseq 39:105 or resseq 107:138 or (resid...B37 - 144
261(chain B and (resseq 39:105 or resseq 107:138 or (resid...B37 - 144
311(chain C and (resseq 39:105 or resseq 107:138 or (resid...C39 - 105
321(chain C and (resseq 39:105 or resseq 107:138 or (resid...C107 - 138
331(chain C and (resseq 39:105 or resseq 107:138 or (resid...C139
341(chain C and (resseq 39:105 or resseq 107:138 or (resid...C37 - 144
351(chain C and (resseq 39:105 or resseq 107:138 or (resid...C37 - 144
361(chain C and (resseq 39:105 or resseq 107:138 or (resid...C37 - 144
411(chain D and (resseq 39:105 or resseq 107:138 or (resid...D39 - 105
421(chain D and (resseq 39:105 or resseq 107:138 or (resid...D107 - 138
431(chain D and (resseq 39:105 or resseq 107:138 or (resid...D139
441(chain D and (resseq 39:105 or resseq 107:138 or (resid...D37 - 144
451(chain D and (resseq 39:105 or resseq 107:138 or (resid...D37 - 144
461(chain D and (resseq 39:105 or resseq 107:138 or (resid...D37 - 144

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Components

#1: Protein
Bacterial proteasome activator


Mass: 15016.304 Da / Num. of mol.: 4 / Fragment: UNP residues 36-159
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: bpa, Rv3780, MTCY13D12.14 / Plasmid: pET24 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: P9WKX3

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.58 Å3/Da / Density % sol: 52.31 %
Crystal growTemperature: 293.15 K / Method: evaporation
Details: 5-7 % (v/v) propane-1,2-diol, 100 mM HEPES-NaOH pH 8.0 - 8.2, 200 mM MgCl2
PH range: 8.0 - 8.2

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 0.9785 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Mar 6, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9785 Å / Relative weight: 1
ReflectionResolution: 3.3→45.35 Å / Num. obs: 16086 / % possible obs: 90.65 % / Redundancy: 9.5 % / Biso Wilson estimate: 95.37 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.11 / Rsym value: 0.116 / Net I/σ(I): 10.4
Reflection shellResolution: 3.3→3.41 Å / Redundancy: 7.68 % / Rmerge(I) obs: 1.001 / Mean I/σ(I) obs: 1.6 / CC1/2: 0.792 / % possible all: 87.9

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Phasing

PhasingMethod: SAD

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Processing

Software
NameVersionClassification
XDSMay 1,2016data reduction
Aimless0.5.26data scaling
SHELXphasing
PHASERphasing
CNSphasing
PHENIX1.10_2155refinement
PDB_EXTRACT3.2data extraction
RefinementMethod to determine structure: SAD / Resolution: 3.303→45.35 Å / SU ML: 0.42 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 38.32
RfactorNum. reflection% reflection
Rfree0.3157 1400 10.04 %
Rwork0.2539 --
obs0.2518 15999 90.24 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 293.46 Å2 / Biso mean: 98.35 Å2 / Biso min: 44.48 Å2
Refinement stepCycle: final / Resolution: 3.303→45.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3324 0 0 0 3324
Num. residues----432
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.013364
X-RAY DIFFRACTIONf_angle_d1.2854564
X-RAY DIFFRACTIONf_chiral_restr0.063544
X-RAY DIFFRACTIONf_plane_restr0.009608
X-RAY DIFFRACTIONf_dihedral_angle_d8.6142872
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A1919X-RAY DIFFRACTION6.868TORSIONAL
12B1919X-RAY DIFFRACTION6.868TORSIONAL
13C1919X-RAY DIFFRACTION6.868TORSIONAL
14D1919X-RAY DIFFRACTION6.868TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 11

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.303-3.40950.38261500.36331249139988
3.4095-3.53140.43351600.3261279143989
3.5314-3.67270.36351620.3071307146991
3.6727-3.83980.35121350.28911290142589
3.8398-4.04220.30211490.25721309145890
4.0422-4.29530.29371360.2331332146892
4.2953-4.62670.29761380.2221335147391
4.6267-5.09180.31811520.23431296144890
5.0918-5.82760.32581380.23461369150793
5.8276-7.33820.32411410.26191360150193
7.3382-49.00650.27361460.19141269141587
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.77030.71381.30246.1388-0.0532.95161.4614-0.86141.12670.8246-0.61591.4773-0.6013-0.36540.20880.5083-0.03630.09121.0790.12181.006515.679631.680723.3535
23.3711.19760.97530.51180.12180.7698-0.7494-0.0644-0.00080.02270.3363-0.87430.32370.20950.00220.80730.0960.08640.6064-0.01040.734619.324223.030916.3908
32.3201-0.30953.64540.14990.0037.95430.280.1366-1.10760.01270.3049-0.46131.43940.23910.53350.8734-0.1447-0.0520.4640.2411.18438.80313.334226.9348
44.1420.76211.1684.7711-0.02982.00080.8634-0.45340.45141.94470.50280.4001-2.7717-0.97340.241.04250.23660.11221.15680.10440.70276.995627.111529.3286
50.854-1.25860.67061.9159-0.62122.3559-0.44130.2878-0.68680.35520.87110.71490.6441-1.31090.0080.6885-0.24020.14250.65680.05580.878927.143143.032523.2525
61.9202-1.0997-0.56281.62941.32921.1682-0.47860.3419-0.7099-0.18140.7154-0.3826-0.2237-0.11530.00120.6553-0.0263-0.00540.70730.11741.105434.357937.401116.4268
70.71682.3665-0.36518.474-3.36087.19220.46580.5088-2.553-0.0722-0.4116-1.8533-0.33841.6316-0.13610.38030.1696-0.06350.8139-0.07660.975230.075123.732926.9444
82.4792-1.08320.66393.36791.09671.07030.1992-0.55280.34452.0038-0.2259-0.0619-0.3413-1.0459-0.17081.651-0.2869-0.11940.8499-0.02370.437421.789634.741529.2604
91.0977-1.326-0.99082.10110.9021.0603-0.4352-0.19990.4940.8234-0.1580.92840.254-0.1429-0.16450.4306-0.16090.03380.6837-0.0460.695930.969458.540523.1462
102.5639-0.3795-0.3450.45150.00810.0539-0.15160.53881.23070.2719-0.8539-0.0592-0.51913.1142-0.06191.01740.0763-0.01790.97910.11040.570740.233157.291716.4179
110.4301-0.2233-0.33331.16190.73511.30381.0385-0.3036-1.00140.6411-0.3814-0.85951.74021.7034-0.00920.72630.0824-0.12860.7191-0.14881.311943.342943.446326.9698
123.495-2.23920.97767.8678-0.62180.2724-0.3499-1.249-0.55192.26041.05050.1783-0.0687-0.78980.42730.4888-0.1096-0.10191.02310.05980.876430.505148.782829.2033
132.90750.1687-1.70982.5196-1.30492.4953-0.0304-0.104-1.99420.09440.71010.90950.8379-0.78390.05060.4619-0.0390.16720.6095-0.08781.044326.789374.019823.3265
142.87651.7272-1.54911.0393-0.85531.1427-0.08170.440.4303-0.726-0.3123-0.18230.07110.75340.00141.02480.2014-0.1830.57310.03961.043535.214877.556716.4507
152.39463.2469-3.06194.4003-4.14933.9165-0.0833-0.77060.36290.4885-0.2767-2.3401-0.01770.9161-2.35180.73390.1002-0.46330.4796-0.16491.220244.816167.027626.9943
162.8692-1.5224-1.88271.19681.70083.7741-0.506-1.32810.19590.99920.72530.22930.9926-1.62320.08431.09450.1956-0.11380.9882-0.21050.661231.415265.442929.2055
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 45 through 63 )A45 - 63
2X-RAY DIFFRACTION2chain 'A' and (resid 68 through 88 )A68 - 88
3X-RAY DIFFRACTION3chain 'A' and (resid 91 through 100 )A91 - 100
4X-RAY DIFFRACTION4chain 'A' and (resid 112 through 144 )A112 - 144
5X-RAY DIFFRACTION5chain 'B' and (resid 45 through 63 )B45 - 63
6X-RAY DIFFRACTION6chain 'B' and (resid 68 through 88 )B68 - 88
7X-RAY DIFFRACTION7chain 'B' and (resid 91 through 100 )B91 - 100
8X-RAY DIFFRACTION8chain 'B' and (resid 112 through 144 )B112 - 144
9X-RAY DIFFRACTION9chain 'C' and (resid 45 through 63 )C45 - 63
10X-RAY DIFFRACTION10chain 'C' and (resid 68 through 88 )C68 - 88
11X-RAY DIFFRACTION11chain 'C' and (resid 91 through 100 )C91 - 100
12X-RAY DIFFRACTION12chain 'C' and (resid 112 through 144 )C112 - 144
13X-RAY DIFFRACTION13chain 'D' and (resid 45 through 63 )D45 - 63
14X-RAY DIFFRACTION14chain 'D' and (resid 68 through 88 )D68 - 88
15X-RAY DIFFRACTION15chain 'D' and (resid 91 through 100 )D91 - 100
16X-RAY DIFFRACTION16chain 'D' and (resid 112 through 144 )D112 - 144

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