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- PDB-5mpp: Structure of AaLS-wt -

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Basic information

Entry
Database: PDB / ID: 5mpp
TitleStructure of AaLS-wt
Components6,7-dimethyl-8-ribityllumazine synthaseLumazine synthase
KeywordsTRANSFERASE / cryo-EM / protein cage / dodecahedron / lumazine synthase
Function / homology
Function and homology information


6,7-dimethyl-8-ribityllumazine synthase / 6,7-dimethyl-8-ribityllumazine synthase activity / riboflavin synthase complex / riboflavin biosynthetic process / cytosol
Similarity search - Function
Lumazine/riboflavin synthase / Lumazine synthase / Lumazine/riboflavin synthase / Lumazine/riboflavin synthase superfamily / 6,7-dimethyl-8-ribityllumazine synthase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSasaki, E. / Boehringer, D. / Leibundgut, M. / Ban, N. / Hilvert, D.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
European Research CouncilERC-dG-2012-321295 Switzerland
CitationJournal: Nat Commun / Year: 2017
Title: Structure and assembly of scalable porous protein cages.
Authors: Eita Sasaki / Daniel Böhringer / Michiel van de Waterbeemd / Marc Leibundgut / Reinhard Zschoche / Albert J R Heck / Nenad Ban / Donald Hilvert /
Abstract: Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of ...Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of two versatile encapsulation systems that exploit engineered electrostatic interactions for cargo loading. We show that increasing the number of negative charges on the lumenal surface of lumazine synthase, a protein that naturally assembles into a ∼1-MDa dodecahedron composed of 12 pentamers, induces stepwise expansion of the native protein shell, giving rise to thermostable ∼3-MDa and ∼6-MDa assemblies containing 180 and 360 subunits, respectively. Remarkably, these expanded particles assume unprecedented tetrahedrally and icosahedrally symmetric structures constructed entirely from pentameric units. Large keyhole-shaped pores in the shell, not present in the wild-type capsid, enable diffusion-limited encapsulation of complementarily charged guests. The structures of these supercharged assemblies demonstrate how programmed electrostatic effects can be effectively harnessed to tailor the architecture and properties of protein cages.
History
DepositionDec 17, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 22, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 17, 2018Group: Data collection / Category: em_image_scans
Revision 1.2Jul 31, 2019Group: Data collection / Refinement description / Category: refine
Revision 1.3Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
A: 6,7-dimethyl-8-ribityllumazine synthase
B: 6,7-dimethyl-8-ribityllumazine synthase
C: 6,7-dimethyl-8-ribityllumazine synthase
D: 6,7-dimethyl-8-ribityllumazine synthase
E: 6,7-dimethyl-8-ribityllumazine synthase
F: 6,7-dimethyl-8-ribityllumazine synthase
G: 6,7-dimethyl-8-ribityllumazine synthase
H: 6,7-dimethyl-8-ribityllumazine synthase
I: 6,7-dimethyl-8-ribityllumazine synthase
J: 6,7-dimethyl-8-ribityllumazine synthase
K: 6,7-dimethyl-8-ribityllumazine synthase
L: 6,7-dimethyl-8-ribityllumazine synthase
M: 6,7-dimethyl-8-ribityllumazine synthase
N: 6,7-dimethyl-8-ribityllumazine synthase
O: 6,7-dimethyl-8-ribityllumazine synthase
P: 6,7-dimethyl-8-ribityllumazine synthase
Q: 6,7-dimethyl-8-ribityllumazine synthase
R: 6,7-dimethyl-8-ribityllumazine synthase
S: 6,7-dimethyl-8-ribityllumazine synthase
T: 6,7-dimethyl-8-ribityllumazine synthase
U: 6,7-dimethyl-8-ribityllumazine synthase
V: 6,7-dimethyl-8-ribityllumazine synthase
W: 6,7-dimethyl-8-ribityllumazine synthase
X: 6,7-dimethyl-8-ribityllumazine synthase
Y: 6,7-dimethyl-8-ribityllumazine synthase
Z: 6,7-dimethyl-8-ribityllumazine synthase
0: 6,7-dimethyl-8-ribityllumazine synthase
1: 6,7-dimethyl-8-ribityllumazine synthase
2: 6,7-dimethyl-8-ribityllumazine synthase
3: 6,7-dimethyl-8-ribityllumazine synthase
4: 6,7-dimethyl-8-ribityllumazine synthase
5: 6,7-dimethyl-8-ribityllumazine synthase
6: 6,7-dimethyl-8-ribityllumazine synthase
7: 6,7-dimethyl-8-ribityllumazine synthase
8: 6,7-dimethyl-8-ribityllumazine synthase
9: 6,7-dimethyl-8-ribityllumazine synthase
a: 6,7-dimethyl-8-ribityllumazine synthase
b: 6,7-dimethyl-8-ribityllumazine synthase
c: 6,7-dimethyl-8-ribityllumazine synthase
d: 6,7-dimethyl-8-ribityllumazine synthase
e: 6,7-dimethyl-8-ribityllumazine synthase
f: 6,7-dimethyl-8-ribityllumazine synthase
g: 6,7-dimethyl-8-ribityllumazine synthase
h: 6,7-dimethyl-8-ribityllumazine synthase
i: 6,7-dimethyl-8-ribityllumazine synthase
j: 6,7-dimethyl-8-ribityllumazine synthase
k: 6,7-dimethyl-8-ribityllumazine synthase
l: 6,7-dimethyl-8-ribityllumazine synthase
m: 6,7-dimethyl-8-ribityllumazine synthase
n: 6,7-dimethyl-8-ribityllumazine synthase
o: 6,7-dimethyl-8-ribityllumazine synthase
p: 6,7-dimethyl-8-ribityllumazine synthase
q: 6,7-dimethyl-8-ribityllumazine synthase
r: 6,7-dimethyl-8-ribityllumazine synthase
s: 6,7-dimethyl-8-ribityllumazine synthase
t: 6,7-dimethyl-8-ribityllumazine synthase
u: 6,7-dimethyl-8-ribityllumazine synthase
v: 6,7-dimethyl-8-ribityllumazine synthase
w: 6,7-dimethyl-8-ribityllumazine synthase
x: 6,7-dimethyl-8-ribityllumazine synthase


Theoretical massNumber of molelcules
Total (without water)1,003,69160
Polymers1,003,69160
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area262650 Å2
ΔGint-1341 kcal/mol
Surface area258660 Å2
MethodPISA

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Components

#1: Protein ...
6,7-dimethyl-8-ribityllumazine synthase / Lumazine synthase / Lumazine synthase


Mass: 16728.186 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus (bacteria) / Gene: ribH, aq_132 / Production host: Escherichia coli (E. coli)
References: UniProt: O66529, 6,7-dimethyl-8-ribityllumazine synthase

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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: AaLS-wt / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Aquifex aeolicus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: na
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 4 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: (1.10-2155_1692: ???) / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND3CTF correction
5RELION1.4CTF correction
11RELION1.4initial Euler assignment
12RELION1.4final Euler assignment
13RELION1.4classification
14RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3268 / Symmetry type: POINT
RefinementResolution: 3.94→252 Å / SU ML: 0.74 / σ(F): 1.99 / Phase error: 24.11 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.2428 13685 2.5 %
Rwork0.2497 --
obs0.2495 546600 99.78 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00971640
ELECTRON MICROSCOPYf_angle_d0.99996780
ELECTRON MICROSCOPYf_dihedral_angle_d14.41442960
ELECTRON MICROSCOPYf_chiral_restr0.05711220
ELECTRON MICROSCOPYf_plane_restr0.00612480

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