DOI: 10.5281/zenodo.13748215
title: An integrative characterisation of proline cis and trans conformers in a disordered peptide

description: These metadynamic molecular dynamics simulations, nuclear magnetic resonance spectroscopy (NMR), and small-angle X-ray scattering (SAXS) data support the findings of the manuscript entitled 'An integrative characterisation of proline cis and trans conformers in a disordered peptide' by Pettitt et al. DOI: 10.1016/j.bpj.2024.09.028

Metadynamic molecular dynamics simulations

The Metadynamic_simulations_Zenodo.tar.xz directory contains data for an N-terminally acetylated disordered peptide. The peptide is the C-terminal region of open reading frame 6 (ORF6-CTR) from SARS-CoV-2. The data were produced by metadynamics simulations in 3 force fields: AMBER03WS (a03ws), AMBER99SB-disp (a99sb), and CHARMM36m (C36m). We used PLUMED version 2.7.1 and GROMCS 2021.2. All the data and PLUMED input files required to reproduce the simulation results are available on PLUMED-NEST. This data should be used with the code provided on GitHub at https://github.com/hansenlab-ucl/orf6-ctr_cis_trans_conformers/tree/master/Metadynamic_simulations

Once downloaded, this directory should be extracted using the following command:

tar -xzvf Metadynamics_simulations_Zenodo.tar.xz

The directory should be saved with the name Metadynamic_simulations_Zenodo and placed in the same directory as the GitHub README_metadynamic_simulations.md file.

This dataset contains:



{system}.pdb - atomic coordinate files for the NAc-ORF6-CTR in the a03ws, a99sb, and C36m force fields.

{system}_traj.trr - single concatenanated trajectory files for the a03ws run 1, a03ws run 2, a99sb, and C36m simulations.

{system}_weights_corr.dat - weights for each frame in _traj.trr for the a03ws run 1, a03ws run 2, a99sb and C36m simulations. Here, the weights of frames in which the peptide interacts with its periodic image have been set to zero. The cutoff was 0.95 nm for C36m, and 1.2 nm for the other force fields.

{system}_weights_saxs_bme.dat - SAXS Bayesian/Maximum Entropy (BME) reweighted weights for each frame in {system}_traj.trr for the a03ws run 1, a03ws run 2, and C36m simulations. Here, the weights of frames in which the peptide interacts with its periodic image have been set to zero. The cutoff was 0.95 nm for C36m, and 1.2 nm for the other force fields.

top_frames_{system}.npy - numpy array of frames index for the a03ws run 1, a03ws run 2, a99sb, and C36m simulations. Frames were selected based on weights_corr.dat.

top_frames_{system}.trr - trajectory of frames for the a03ws run 1, a03ws run 2, a99sb, and C36m simulations. Frames were selected based on weights_corr.dat

top_frames_{system}_rw_cis.npy - numpy array of cis-P57 frames index for the a03ws run 1, a03ws run 2, and C36m simulations. Frames were selected based on _weights_saxs_bme.dat

top_frames_{system}_rw_trans.npy - numpy array of trans-P57 frames index for the a03ws run 1, a03ws run 2, and C36m simulations. Frames were selected based on _weights_saxs_bme.dat

top_frames_{system}_rw_cis.trr - trajectory of cis-P57 frames for the a03ws run 1, a03ws run 2, and C36m simulations. Frames were selected based on _weights_saxs_bme.dat

top_frames_{system}_rw_trans.trr - trajectory of trans-P57 frames for the a03ws run 1, a03ws run 2, and C36m simulations. Frames were selected based on _weights_saxs_bme.dat

CS_COLVAR_{system} - experimental and CamShift predicted chemical shifts for all four systems (a03ws run1, run2, a99sb, and C36m) as well as the cis and trans sub-ensembles for a03ws run1, a03ws run2, and C36m


Nuclear magnetic resonance spectroscopy (NMR) data

The NMR_Zenodo.tar.xz directory contains data for the backbone and sidechain assignment experiments, backbone 15N relaxation rates experiments, and 15N diffusion experiments (.ft2 and .ft3 files). Unless specified otherwise, NMR spectra were collected on uniformly 15N-labelled and 13C, 15N-labelled ORF6-CTR at concentrations of 300 micromolar and on unlabelled NAc-ORF6-CTR at concentations of 400 micromolar. All peptides were prepared in 25 mM HEPES buffer at pH 6.9, 150 mM NaCl, containing 5% D2O, 1 mM sodium azide, and 1 mM EDTA. NMR data were recorded at 288.15 K (15 degrees celsius). Spectra were measured at a static magnetic field strength of 14. T (600 MHz), unless specified otherwise.

Once downloaded, this directory should be extracted using the following command:

tar -xzvf NMR_Zenodo.tar.xz

Note: this data is not required to run the NMR analysis on GitHub at https://github.com/hansenlab-ucl/orf6-ctr_cis_trans_conformers/tree/master/NMR but it can be used for you to repeat your own analysis.

NMR chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank (BMRB; www.bmrb.wisc.edu) under the following accession codes: 52459 for the ORF6CTR cis-P57 and trans-P57 configurations, and 52460 for the unlabelled NAc-ORF6CTR.

This dataset contains:



1H-1H-tocsy-nac-orf6-ctr.ft2 - 2D 1H-1H tocsy on the NAc-ORF6-CTR. Standard dipsi2esgpph Bruker pulse sequence, with spectral widths of 14 ppm (direct dimension) and 12 ppm (indirect dimension), and 1,024 x 512 complex points, respectively. 32 scans were recorded, with an inter-scan delay of 1.5 s.

1H-1H-tocsy.ft2 - 2D 1H-1H total correlation spectroscopy (TOCSY) on the ORF6-CTR. Same details as above.

1H-13C-hsqc-nac-orf6-ctr.ft2 - 2D 1H-13C heteronuclear single quantum coherence (HSQC) spectra on the NAc-ORF6-CTR. Standard hsqcgpph Bruker sequence was employed, with 192 scans, spectral widths of 14 ppm (1H) and 80 ppm for (13C), and 512 x 180 complex points, respectively. The recycle delay was set to 1.5 s, with carriers set to the position of the water peak (1H) and 35 ppm relative to TMS (13C).

1H-15N.ft2 - 2D 1H-15N HSQC spectra on the 15N-labelled ORF6-CTR. Spectra were acquired using the hsqcetf3gpsi2 Bruker pulse sequence. Spectral widths were set to 16 ppm (1H) and 20.5 ppm (15N), with 1,536 x 128 complex points acquired, respectively. Carriers were set to the position of the water peak (1H) and 121.5 ppm (15N), with 4 scans and a recycle delay of 1 s.

1H-15N-hsqc-310K.ft2 - 2D 1H-15N HSQC spectra on the 15N-labelled ORF6-CTR at 310.15 K (37 degrees celsius). Same details as above.

15N-edited-dosy-950MHz.ft2 - Pseudo-3D diffusion pulsed field-gradient spin echo (PFGSE) 1H-15N-Diffusion Ordered Spectroscopy-HSQC (1H-15N-DOSY-HSQC) experiment with a bipolar gradient at a static magnetic field strength of 22.3 T (950 MHz) on the 15N-labelled ORF6-CTR. Six gradient experiments were acquired for each data set, with the gradient strengths augmented linearly through the acquisition from 0.9 to 39.5 G/cm and all other delays and pulses held constant. Gradient pulses (δ) were applied for 3 ms and a diffusion delay (Δ) of 200 ms. 80 scans were acquired per gradient experiment with 1,536 x 176 complex points (1H, 15N), using the same spectral widths and carriers as used before for the 2D 1H-15N HSQC experiment.

15N-edited-noesy.ft3 - 15N-edited nuclear Overhauser effect spectroscopy HSQC (15N-NOESY-HSQC) on the ORF6-CTR. Spectra were acquired using the noesyhsqcfpf3gpsi3d Bruker pulse sequence. Spectra were recorded with 8 scans, 2,048 x 24 x 96 complex points (1H, 15N, 1H), and a spectral width of 16 x 20.5 x 16 ppm, respectively. Carriers were set to the water peak (1H) and 121.5 ppm (15N). A recycle delay of 1.5 s and a mixing time of 0.25 s were used

15N-edited-tocsy.ft3 - 15N-TOCSY-HSQC on the ORF6-CTR. Spectra were acquired using a pulse sequence based on the original Bruker dipsihsqcf3gpsi3d with a flip-flop spectroscopy (FLOPSY)-16 scheme. Spectra were acquired with 16 scans, 1,024 x 32 x 96 complex points (1H, 15N, 1H), and a spectral width of 16 x 20.5 x 16 ppm, respectively. Carriers were set to the water peak (1H) and 121.5 ppm (15N), with a recycle delay of 1 s. A TOCSY mixing time of 0.1 s was used and an 8 kHz TOCSY spin lock was applied.

ccconh.ft3 - CC(CO)NH spectra were acquired on the 13C, 15N-labelled ORF6-CTR. Spectra were acquired using a pulse sequence based on the original Bruker ccconhgp3d.2 with a FLOPSY-16 scheme. Spectra were recorded with 16 scans, 1,024 x 30 x 84 complex points (1H, 15N, 13C), and a spectral width of 14 x 20.5 x 71 ppm, respectively. Carriers were set to the water peak (1H), 121.5 ppm (15N) and 43 ppm (13C). A recycle delay of 1.5 s, along with a TOCSY mixing time of 18 ms were used.

con.ft2 - 2D CON spectra were acquired on the 13C, 15N-labelled ORF6-CTR. Spectra were acquired using the c_con_iasq Bruker pulse sequence. Spectral widths were set to 40 ppm in both dimensions, with carriers set to 173 ppm relative to TMS (13C) and 121.5 ppm (15N), and 512 x 128 complex points, respectively. 32 scans and a recycle delay of 2 s were applied.

hncacb.ft3 - The 3D HNCACB spectra were acquired on the 13C, 15N-labelled ORF6-CTR using the hncacbgp3d Bruker pulse sequence. Spectra were recorded with 16 scans, 1,024 x 30 x 72 complex points (1H, 15N, 13C), and a spectral width of 14 x 20.5 x 60.2 ppm, respectively. Carriers were set to the water peak (1H), 121.5 ppm (15N), and 43 ppm (13C), with a recycle delay of 1 s.

hncaco.ft3 - The 3D HN(CA)CO spectra were acquired using the hncacogpwg3d Bruker pulse sequence on the 13C,15N-labelled ORF6-CTR. Spectra were recorded with 16 scans, 1,024 x 29 x 80 complex points (1H, 15N, 13C), and a spectral width of 14 x 20.5 x 7 ppm, respectively. Carriers were set to the position of the water peak (1H), 121.5 ppm (15N) and 173 ppm relative to TMS (13C), with a recycle delay of 1 s.

hnco.ft3 - The 3D HNCO spectra were acquired using the hncogp3d Bruker pulse sequence on the 13C, 15N-labelled ORF6-CTR. The same details were used as for the hncaco.ft3.

noe.ft2 - The {1H}-15N hetNOEs were recorded using a pseudo-3D experiment, with and without proton saturation on the 15N-labelled ORF6-CTR. Amide proton magnetisation saturation was accomplished by using a 5 s train of high-power 120° pulses applied at 5 ms intervals. The reference and saturated spectra were alternately recorded. To ensure complete recovery of the initial magnetisation at the start of each increment of the reference experiment, a long recycle delay of 15 s was applied. The spectral widths were 16 ppm (1H) and 20.5 ppm (15N), and 2,048 x 160 (1H, 15N) complex points were recorded at a static magnetic field strength of 14.1 T.

noe-800MHz.ft2 - The same details as for noe.ft2, except it was measured at a static magnetic field strength of 18.8 T (800 MHz).

r1.ft2 - Rates were measured using established proton-detected pulse sequences based on a gradient-selected, sensitivity-enhanced, refocused 15N sequences on the 15N-labelled ORF6-CTR. Spectra were recorded with 2,048 x 160 complex points and spectral widths as used before in the 2D 1H-15N HSQC experiment. Gradient pulses were used to suppress the water signal and a long recycle delay of 3 s was employed. N-H cross-correlated relaxation pathways were suppressed by hard 180˚ pulses every 20 ms during the relaxation delay. The R1 N-H planes were recorded with 8 relaxation delays ranging from 20 ms to 700 ms.

r1-800MHz.ft2 - Same details as for r1.ft2, except the rates were measured at a static magnnetic field strength of 18.8 T (800 MHz).

r1rho.ft2 - Similar details to r1.ft2, except cross-correlated relaxation was suppressed by hard 180˚ pulses during the 15N spin-lock. Magnetisation was explicitly aligned with the spin-lock field. N-H planes were recorded using 8 relaxation delays ranging from 2 ms to 140 ms, with a 15N spin-lock field strength of 2 kHz.

r1rho_800MHz.ft2 - Same as r1rho.ft2, except the rates were measured at a static magenetic field strength of 18.8 T (800 MHz).

rdd.ft2 - Exchange-free 15N transverse relaxation (Rdd) rates were measured using pulse schemes for the four 1H-15N relaxation rates R1ρ(2HzN’z), R1ρ(2H’zNz), R1ρ2(2H’zN’z), and R1(2HzNz) on the 15N-labelled ORF6-CTR. A total of 8 relaxation delays between 2 ms and 26 ms were used for all experiments (same delays used for each rate measurement). A 10 kHz 1H spin lock and a 2 kHz 15N spin lock were applied. Spectral widths were the same as those used before in {1H}-15N hetNOE experiments, with 1,536 x 640 (1H, 15N) complex points.


Small-angle X-ray scattering (SAXS) data

Experimental and predicted SAXS data for the NAc-ORF6-CTR. Once downloaded, this directory should be extracted using the following command:

tar -xzvf SAXS_calculations_Zenodo.tar.xz

This dataset contains:



saxs_exp_nac_orf6_ctr.dat - experimental SAXS data collected on Instrument B21 at Diamond Light Source (Didcot, UK). Measurements were recorded on 800 micromolar unlabelled ORF6-CTR at 310.15 K (37 degrees celsius). Data sets of 26 frames with a frame exposure time of 1 s each were acquired.

{system}_saxs_calc_nac_orf6_ctr.dat - calculated SAXS data using PEPSI-SAXS for the a03ws run 1, a03ws run 2, and C36m metadynamics simulations. There is one column per experimental average (total = 2570) and a row for each frame in the trajectory, which depends on the number of frames in the a03ws run 1, a03ws run 2, and C36m simulations.


Jupyter notebooks

All Jupyter notebooks can be accessed from GitHub. Open Jupyter notebooks using jupyter lab in the analysis environment.