Structural Analysis of the Partially Disordered Protein EspK from Mycobacterium Tuberculosis

Gijsbers A, Sánchez-Puig N, Gao Y, Peters P, Ravelli R, Siliqi D, Crystals 11(1):18 (2020) DOI

SASDKQ4 – EspK secretion protein - full length

ESX-1 secretion-associated protein EspK
MWexperimental 95 kDa
MWexpected 27 kDa
VPorod 330 nm3
log I(s) 1.54×102 1.54×101 1.54×100 1.54×10-1
ESX-1 secretion-associated protein EspK small angle scattering data  s, nm-1
ln I(s)
ESX-1 secretion-associated protein EspK Guinier plot ln 1.54×102 Rg: 7.2 nm 0 (7.2 nm)-2 s2
(sRg)2I(s)/I(0)
ESX-1 secretion-associated protein EspK Kratky plot 1.104 0 √3 sRg
p(r)
ESX-1 secretion-associated protein EspK pair distance distribution function Rg: 0.9 nm 0 Dmax: 3.3 nm

Data validation

Fits and models

log I(s)
 s, nm-1
ESX-1 secretion-associated protein EspK CORAL model
log I(s)
 s, nm-1
ESX-1 secretion-associated protein EspK DAMFILT model
The structural characterisation of the EspK full length protein was performed by SAXS coupled to an online size exclusion chromatography equilibrated with 20 mM Tris-HCl pH 8.0, 300 mM NaCl. The SAXS were collected in the bioSAXS beamline P12-EMBL at DESY Light Source, Hamburg, Germany. Sample consisted of 50 µL at a concentration of 3.6 mg mL-1 and was injected onto a Superdex 200 Increase 3.2/300 size exclusion column attached to a FPLC–Malvern TDA system at a flow rate of 0.1 mL min-1. The elution output was directed through a quartz capillary cell (50 µm thick wall and a 1.7 mm path length) held in vacuum. Data acquisition consisted of 1850 frames (with 1 s exposure time) using a PILATUS 2M detector at the distance of 3.0 m from the sample. Collected two-dimensional images were corrected for variations in beam current, normalized for time exposure, and processed into one-dimensional scattering curves using integrated software at the beamline. Background was manually subtracted using the program Chromix (ATSAS). The first atomic model as obtained by fitting, by ChimeRA, the three part of the protein , obtained by ITASSER, into low resolution SAXS DAMMIF model. This initial model was next refined by Coral, adding the linkers between the middle part with C and N-terminal

ESX-1 secretion-associated protein EspK (EspK(484-729))
Mol. type   Protein
Organism   Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Olig. state   Monomer
Mon. MW   27.2 kDa
 
UniProt   P9WJC1 (484-729)
Sequence   FASTA