DOI: 10.15125/bath-01066
title: Dataset for "Ab initio reconstruction of small angle scattering data for membrane proteins in copolymer nanodiscs"

description: The purpose behind this dataset is to investigate the use of form-factor based SANS fitting methods and compare them to dummy-models generated using the ab initio modelling tools. The paper focusses on two different membrane proteins, outer membrane protein F (OmpF) and gramicidin incorporated into polymer nanodiscs with styene maleic acid (SMA). These constructs were compared to other techniques (DLS and CD) to discuss the differences that arise from investigating these complex systems using various methods. These were also compared to known crystal structures. 

This dataset holds the raw data files for the paper titled: "Ab initio reconstruction of small angle scattering data for membrane proteins in copolymer nanodiscs". Techniques include; size exclusion chromatography (SEC) for purification prior to other techniques; circular dichroism (CD); dynamic light scattering (DLS); small angle neutron scattering (SANS) and ab initio modelling constructs.
description: Samples were generated as described in "Ab initio reconstruction of small angle scattering data for membrane proteins in copolymer nanodiscs". In summary;

Gramicidin sample preparation: 
A stock of 20 mM DMPC with 320 µM gramicidin liposomes were prepared and solubilised using SMA. Final concentrations were 10 mM DMPC, 160 µM gramicidin and 2.5 % (w/v) SMA. 

OmpF enrichment:
Endogenous OmpF enrichment was adapted from previous literature (Efremov and Sazanov, 2012). E.coli cells are homogenised and broken, and membranes are harvested. Membranes were incubated with 1% (w/v) Triton X-100 for 15 minutes before ultracentrifugation. The resulting pellet was resuspended and incubated with 2% (w/v) Triton X-100 for 10 minutes before ultracentrifugation. The resulting pellet was solubilised with 2.5 % (w/v) SMA  for 1 hour prior to ultracentrifugation, and the supernatant containing soluble OmpF-nanodiscs was collected. Excess polymer and aggregates were removed by size exclusion chromatography (SEC). Purified samples underwent lipid-exchange and buffer exchange for SANS and CD experimentation. 

Lipid exchange:
To obtain lipid contrasts for SANS datasets, samples underwent lipid-exchange with deuterated-DMPC (d-DMPC) nanodiscs (10 mM d-DMPC with 1.5 % (w/v) SMA). Samples were then purified using SEC to remove excess polymer, followed by concentration using centrifugal filters. 

Circular Dichroism:
Circular dichroism (CD) spectra were measured between 260 – 185 nm at 20°C using a 1.00 mm quartz cuvette using a Chirascan CD spectrometer (Applied Photophysics, UK) with the protein sample at 0.2 mg/mL for CD analysis. DMPC-only nanodiscs were used as a control. To remove any ‘background’ interference from the CD spectrum, the signal from the DMPC-only nanodisc sample was subtracted from that from the OmpF samples. The data was analysed using Dichroweb to determine the nature of the protein structure within the nanodiscs.

SANS:
Measurements were made on the SANS2d instrument at ISIS Neutron and Muon source (Harwell, UK) using 1mm thick quartz Hellma cells and placed in a temperature-controlled sample holding rack at 25C. Three contrasts were used for gramicidin; d-DMPC with hydrogenated-PBS (h-PBS), d-DMPC with 32% deuterated-PBS (d-PBS) and hydrogenated-DMPC with d-PBS; OmpF contrasts were partially deuterated bacterial lipids (d-bact) with h-PBS, d-bact with 32% d-PBS and d-bact with d-PBS. 

Dynamic light scattering:
All DLS measurements were performed using a Malvern Panalytical Zetasizer Nano ZS. Size distribution was measured and is shown on a log scale, plotted using Igor Pro 6 (Wavemetrics) software.
description: CD:
Background was subtracted from CD data using lipid-only nanodiscs to remove any polymer inference. The data were analysed using Dichroweb and compared using predicted CD spectra for OmpF crystal structure (PDB ID: 2OMF), determined by PDBMD2CD. 

SANS:
Data were corrected, solvent subtracted and reduced to 1-D curves using Mantid software.

Gramicidin nanodisc SANS data ab initio modelling:
To create an overall shape of the gramicidin nanodisc, DAMMIF was used before further refinement using;  DAMSEL, DAMSUP, DAMAVER, DAMFILT, and DAMSTART. Lastly, the models were passed through DAMMIN, to compare against the original data file. 

OmpF nanodisc SANS data ab initio modelling:
A multi-phase construct was generated using MONSA for the OmpF-nanodiscs. Further refinement was conducted for each phase separately using; DAMSEL, DAMSUP, DAMAVER, DAMSTART and DAMFILT. The two refines phases were then superimposed using SUPCOMB to correct their orientation to one another.
description: CD:
CD spectra obtained by a Chirascan CD spectrometer (Applied Photophysics, UK). Data was analysed using DICROWEB web interface and compared to predicted CD spectra from crystal structures (PDB ID: 2OMF) using PDBMD2CD web interface. Data was visualised using Igor Pro 6 (Wavemetrics) software. 

DLS:
DLS measurements were performed using a Malvern Panalytical Zetasizer Nano ZS and visualised using Igor Pro 6 (Wavemetrics) software. 

SANS:
SANS data obtained by SANS2d instrument at ISIS Neutron and Muon source (Harwell, UK) and reduced to 1-D curves using Mantid software. Resulting datasets can be visualised using Igor Pro 6 (Wavemetrics) and fitted using cylindrical nanodisc models (part of the SANS analysis software developed at NIST, modified to incorporate the water content in the shell).

Ab initio model constructs:
Initial models are generated using Data analysis software (ATSAS). For gramicidin nanodiscs, DAMMIF, DAMSEL, DAMSUP, DAMAVER, DAMFILT, DAMSTART and DAMMIN, were used to refine and generate the final model construct against the original data file. These create a .pdb file which can be visualised using Visual Molecular Dynamics (VMD) software. For OmpF nanodiscs, MONSA, DAMSEL, DAMSUP, DAMAVER, DAMSTART, DAMFILT and SUPCOMB were used to refine, generate the final model construct against the original data file and to correct the multiple phase orientations to one another. These create a .pdb file which can be visualised using Visual Molecular Dynamics (VMD) software.
description: Details of the data and labelling is available in the README.txt file.