Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis.

Thongkawphueak T, Winter AJ, Williams C, Maple HJ, Soontaranon S, Kaewhan C, Campopiano DJ, Crump MP, Wattana-Amorn P, Biochemistry (2021) Europe PMC

SASDJ59 – Holo PigH ACP1-ACP2

4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH
MWexperimental 31 kDa
MWexpected 22 kDa
VPorod 72 nm3
log I(s) 5.37×10-2 5.37×10-3 5.37×10-4 5.37×10-5
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH small angle scattering data  s, nm-1
ln I(s)
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH Guinier plot ln 5.38×10-2 Rg: 2.8 nm 0 (2.8 nm)-2 s2
(sRg)2I(s)/I(0)
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH Kratky plot 1.104 0 √3 sRg
p(r)
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH pair distance distribution function Rg: 2.8 nm 0 Dmax: 9.7 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Holo PigH ACP1-ACP2 Rg histogram Rg, nm
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH EOM/RANCH model
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH EOM/RANCH model
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH EOM/RANCH model
log I(s)
 s, nm-1
Holo PigH ACP1-ACP2 Rg histogram Rg, nm
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH EOM/RANCH model
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH EOM/RANCH model
4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH EOM/RANCH model
Synchrotron SAXS data from solutions of Holo PigH ACP1-ACP2 in 25 mM Tris, 150 mM NaCl, pH 7.5 were collected on the B21 beam line at the Diamond Light Source storage ring (Didcot, UK) using a Pilatus 2M detector at a sample-detector distance of 4 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 45.00 μl sample at 13 mg/ml was injected at a 0.07 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 25°C. 620 successive 3 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Synchrotron SAXS data from solutions of holo PigH ACP1-ACP2 25 mM Tris 150 mM NaCl, pH 7.5 were collected using in-line SEC-SAXS at the B21 beam line at the Diamond Light Source (Oxfordshire, UK) using a Pilatus 2M detector at a sample-detector distance of 4.0 m and at a wavelength of λ = 0.1 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In line SEC-SAXS was performed using an Agilent 1200 HPLC system equipped with a 2.4 mL Superdex S200 column (GE Healthcare). A sample concentration of 13 mg/ml was loaded onto the column at a flow rate of 0.075 mL/min. Data frames were collected at 25°C for the entire column elution with an exposure time of 3 seconds per frame through an angular s-range of 0.04-3.5 nm-1. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted using data frames measured from the flow-through column buffer with no macromolecular sample present. The model fits, Rg distributions and a sub-set of ensemble model representatives as shown described: Top: EOM of holo PigH ACP1-ACP2 when modelling R83-S99 as flexible. Bottom. EOM of holo PigH ACP1-ACP2 when modelling S88-S99 as flexible For clarity, only 3 ensemble model representatives from the combined pool are shown. The complete EOM-refined model-pool representatives and EOM log files are made available in the full entry zip archive.

4-hydroxy-2,2'-bipyrrole-5-methanol synthase PigH (PigH ACP12)
Mol. type   Protein
Organism   Serratia sp.
Olig. state   Monomer
Mon. MW   22.2 kDa
 
UniProt   Q5W264 (1-189)
Sequence   FASTA