Tutorial

Tutorial

Table of contents

SMD (steered molecular dynamics) – method description

Steered molecular dynamics is a non-equilibrium molecular dynamics method ideal for studying the resistance encountered by a molecule when it is moved in a certain direction. The resistance experienced by the molecule is expressed as a force required to overcome it. Speaking more precisely, a virtual atom moving with a constant velocity (the velocity value and its vector are specified by the user) is connected to the real atom (ie. SMD atom)* of the studied molecule by a virtual spring (spring force constant value and the SMD atom are also specified by the user). The force is applied to the virtual atom, its value adjusted automatically in order to maintain the constant velocity of the virtual atom. The force value and the coordinates of SMD atom are monitored and recorded throughout the simulation. The data recorded during a SMD simulation allow for calculation of the energy barriers encountered by the molecule during its journey (). By sampling different pulling directions it is possible to explore the degrees of freedom of a molecule in a studied system as well as to obtain the reaction coordinates that can be used for umbrella sampling (another sampling method useful for mapping the energy landscape of a studied system).

* In GS-SMD the Cα atom of the C-terminal residue of the substrate is defined as an SMD atom.

Force
Work

Plots based on SMD simulations showing force and work/energy vs. time. Results from four simulations with identical SMD parameters of the same system GS-APP (γ-secretase-amyloid precursor protein) are shown on each figure.

Example of analysis of single SMD trajectory (trajectory in red in F/t plot)

Secondary structure

By analyzing the trajectory the user can identify the hydrogen bonds that are creating/breaking during the simulation. The linear plots show distances between the interacting atoms from two selected residues at C-terminus of APP (1-33) substrate (Val28-Thr32 in orange, and Ile29-Thr32 in red). The plots indicate presence of hydrogen bond between side chain of Thr32 and carbonyl group of Val28 and then Ile29, That bond hopped from residue 28 to 29 at 3 ns, and was broken at about 14 ns - that event is associated with significant force drop in F/t plot. The same events can be seen in HeatMap (the interactions in the above residue pairs are encircled) together with other hydrogen bond creating/breaking events between other residues. The presented HeatMap shows frequencies [0-1] of hydrogen bonds within APP substrate for different time intervals. Since such analysis is highly dependent on GS-substrate/mutant pair, it is not part of the server and should be done on individual basis.

In order to submit your own input for a simulation press the button or click the “MD&mut” label at the top action bar.
A “Molecular dynamics & mutations” window pops out.

Timeline

In this page the structures of γ-secretase complex deposited in Protein Data Bank (PDB) can be seen. All structures deposited so far are available on a timeline. By selecting the PDB ID the structure of γ-secretase shows up in a window and one can visualize its structure using options from the control panel. The details of citation with a DOI link are shown above the structure window.

0

Molecular dynamics & mutations

By clicking the button in order to fill the whole ‘Molecular dynamics & mutations’ form with a sample input data.

In the field number 1 labeled ‘γ Secretase Mode’ the user selects how large fragment of the γ-secretase complex should be included in the simulations (appropriate protein fragment is highlighted when clicked)

  • All – the whole γ-secretase complex: Nicastrin (chain A), residues 34-700; Presenilin-1 (chain B), residues 73-298, 374-467; Aph1-A (chain C), residues 2-244; PEN-2 (chain D), residues 2-101 are included in the simulation
  • Membrane part – only transmembrane part of the γ-secretase complex: transmembrane helix of Nicastrin (chain A), residues 664-700; Presenilin-1 (chain B), residues 73-298, 374-467; Aph1-A (chain C), residues 2-244; PEN-2 (chain D), residues 2-101 are included
  • Presenilin – only the catalytic subunit of γ-secretase, Presenilin-1 (chain B), residues 73-298, 374-467 is included (the long loop between transmembrane helices TM6 and TM7 is not determined)

2

In the field nr. 2 labeled ‘Substrate’ the user specifies what substrate is going to be used in the SMD simulation. In the upper line (‘Peptide substrate selection’) the initial version of substrate peptide (a template for sequence threading) is selected from a pop-up menu which appears when the line is clicked. In the lower line (‘Sequence for threading to substrate structure’) the sequence to be threaded is specified by the user. The cleavage point (marked by ‘*’) is used as a reference position for a sequence threading. The final sequence for the simulation is highlighted in red below the lower line. The final sequence should be between 15 and 33 AA long. This sequence will be threaded to the substrate structure in PDB ID:6IYC γ-secretase.

3

Here is the list of Selected substrates of γ-secretase with known cleavage point (available in Substrate input)

Protein NameUniProt IDMembrane region sequence
Alcadein α (Calsyntenin-1)O94985|CSTN1_HUMAN
Q9EPL2|CSTN1_MOUSE
VVPSTAT*VVIVVCVSFLVFMIIL*GVFRIRAA
Alcadein β (Calsyntenin-3)Q9BQT9|CSTN3_HUMANMIPSAATLII*VVCVGFLVLMVVLG*LVRIHSLH
Alcadein γ (Calsyntenin-2)Q9H4D0|CSTN2_HUMANSVVPSIAT*VVIIISVCMLVFVVAM*GVYRVRIA
APLP1Q03157|APLP1_MOUSE
P51693|APLP1_HUMAN
GVSREAVSGLLIMGAGG*GSLIVL*SMLLLRRKKP
APLP2Q06481|APLP2_HUMAN
Q06335|APLP2_MOUSE
FSLSSSA*LIGL*LVIAVAIATVIVISL*VMLRKRQY
APPP05067|A4_HUMANVGSNKGAIIGLMVGGVV*IATVIVITL*VMLKKKQY
CD44P16070|CD44_HUMANPQIPEWLIILASLLA*LALILAVCI*AVNSRRR
CSF-1RP09581|CSF1R_MOUSESLFTPVVVACMSVMSLLVLLLL*LLLYKYKQK
E-CadherinQ90762|CADH6_CHICK
P09803|CADH1_MOUSE
P12830|CADH1_HUMAN
P19022|CADH2_HUMAN
P15116|CADH2_MOUSE
P33151|CADH5_HUMAN
LQIPAILGILGGILALLILILLLLLFL*RRRRA
EpCAMP16422|EPCAM_HUMANMQGLKAGVIAVIV*VVVIAVVAGIV*VLVISRKKR
EphB2P54763|EPHB2_MOUSEEKLPLIVGSSAAGLVFLIAVVVI*AIVCNRRG
GHRP16882|GHR_MOUSE
P19941|GHR_RABIT
EEDFRFPWFLIIIFGIFGLTVMLFV*FIFSKQQRI
IL-1R2P27930|IL1R2_HUMANTTVKEASSTFSWGIV*LAPLS*LAFLVLGGIWMHRRCK
Neuregulin-1Q6DR98|Q6DR98_MOUSELYQKRVLTITGICIAL*LVVGIMC*VVAYCKTKKQ
Notch-1Q01705|NOTC1_MOUSEPAQLHFMYVAA*AAFVLLFFVGCG*VLLSRKRR
Notch-2O35516|NOTC2_MOUSEPERTQLLYLLAVAVV*IILFIILLG*VIMAKRKRK
Notch-3Q61982|NOTC3_MOUSEPSVPLLPLLVA*GAVLLLVILVLG*VMVARRKRE
Notch-4P31695|NOTC4_MOUSEANQLPWPVLCSPVAG*VILLALGALL*VLQLIRR
p75 NTRP08138|TNR16_HUMANGTTDNLIPVYCSILAAVV*VGLVAYIAFKRWNS
Podoplanin (PDPN)Q86YL7|PDPN_HUMANSTVTLVGIIVGVLLAIGFIGAIIV*VVMRKMS
PTK7Q13308|PTK7_HUMANKMIQTIGLSVGAAVAYIIAVLG*LMFYCKKRC
Syndecan-3O75056|SDC3_HUMAN
Q64519|SDC3_MOUSE
ERKEVLVAVIVGGVVGALFAAL*VTLLIYRMKK
VEGFR-1 (FLT1)F1MDD9|F1MDD9_BOVINKSNLELITLTCT*CVAATLFWLLL*TLFIRKMKR

A complete list of all substrates can be found in a recent publication https://doi.org/10.1016/j.semcdb.2020.05.019

The ‘*’ sign indicates the experimentally identified cleavage position/s in all sequences.

In the field nr. 3 labeled ‘SMD’ the parameters for SMD simulation (such as: SMD velocity in [m/s], virtual spring force constant in [kcal/mol/Å2], the x,y,z coordinates of the force vector in [Å]) are provided by the user. One can pull not only the last residue (33) of the substrate but also any set of substrate residues (chain E) by specifying them in the 'SMD residues list' field.

4

Magnification of the structure showing the pulling direction selected by user (yellow arrow).

arrow MD

sets all the SMD parameters to their default values

To the right is the visualization of gamma-secretase with a substrate. The membrane opacity can be adjusted using the slider above. The user can navigate the visualization state with the four buttons below:

- centers the whole complex

- toggles the full screen view (press ‘esc’ to exit the full screen mode)

- changes the background color from black to white or from white to black

In the field nr. 4 labeled ‘γ-Secretase Mutations’ the user can introduce specific mutations into the γ-secretase complex that is going to be used in the simulation. Each line stands for a different subunit of the enzyme. Mutations should be specified using one letter code and a following format: AnB where “A” is the residue to be mutated, “N” is the residue number and “B” is a destination residue. For instance if you want to mutate histidine 100 to alanine you should type “H100A”

5

In the field nr. 5 labeled ‘Dynamics’ the user can set the length of the simulation (Total length [ns], default value is 10), the number of frames in the trajectory (number of frames, default value is 100, please note that the more frames you decide to save the heavier the output files will be) and the implicit membrane thickness (Membrane thickness [Å], default value is 31.0).

6

After adjusting all the parameters click to start the SMD simulation.

If you wish to set all the parameters to their default values, click


Examples

In order to see some sample results you should click ‘Example1’ and ‘Example2’ at the top menu.

Example 1: pulling of C-terminus (Cα atom of residue 33) of APP in all γ-secretase structure.

Example 2: pulling of N-terminus (residues 1-21) of APP in PS-1 structure. An arrow indicates pulling of center of mass of selected residues (Cα atoms).


Analysis of MD

7

Magnification of the structure showing the pulling direction (yellow arrow) and the distance of the virtual atom to SMD atom (in red).

arrow smd
Downloads

Download the trajectory package as zip file containing trajectory (in DCD format) and the files for making flare plots and frequency analyses (button: above the flare plot)

Download the current view of the flare plot (button above the flare plot)

Download the current view of the structure visualization (button below the trajectory visualization window)

Flareplot control

Interaction type selection – below the flareplot there is pop-up-on-click-menu where the user can select the interaction types to be visualized (such as: salt bridges, hydrogen bonds, aromatic, VdW or all types at once). The interactions of the substrate to the subunits of γ-secretase, as well as internal interactions in the substrate are displayed.

Slider (frame/frame range) selection – using the slider below the flareplot window the user can select the frame for which the interactions should be visualized. In addition the slider is synchronized with a NGL visualization of the trajectory, therefore the user can simultaneously track the structural changes of the system and the changes in the interaction network. The slider allows for selection of a range of frames as well and in such case the interactions are shown for more than one frame (the line thickness is proportional to the frequency of the particular interaction within a selected range of frames) and the NGL visualization show the first frame from the selection.

NGL control

  • Membrane opacity - The slider on NGL window – allows to adjust the opacity of the membrane representation (blue and red surfaces representing the membrane borders)
  • - centers the structure
  • - activates the full screen view (to exit the full screen press ‘Esc’)
  • - activate a slow rotation (spinning) of the system
  • - restores default view settings
  • Representation – a pop-up menu where the user can choose a representation of the structure (such as: cartoon, backbone, surface, etc.)
  • Figure quality – a pop-up menu where the user can adjust the quality of the visualization (affects also the quality of the image saved when using described in ‘Downloads’ section)
  • Color – a pop-up menu where the user can select the coloring scheme for the structure visualization. Available coloring methods are: GS-SMD server, which is coloring by protein chain; rainbow
  • View mode - a pop-up menu allowing to switch between perspective and orthographic view
8

Charts

Below the flareplot and NGL visualization the plots of Force in a function of Time and of Energy/Work in a function of Time are shown. Both plots are based on SMD simulations results

Force
Work

SMA - Simple Moving Average

Simple Moving Average (SMA) uses a sliding window to take the average over a set number of time periods. It is an equally weighted mean of the previous n data points.

The Secondary structure plot together with Job information

H - α-Helix, G - 310-Helix, I - π-Helix, E - Strand, B - Bridge, C - Coil, T - Turn.


Shared jobs

Clicking the ‘Shared jobs’ leads to a GS-SMD server trajectories data base. The trajectories from MD simulations performed by the users who agreed for their results to be available for public are kept here. In order to submit your results to the ‘Shared jobs list’ on the results page click button.

10

NGL mouse controls

Left button hold and move to rotate around center

Left button click to pick an atom

Middle button click to center on atom

Middle button hold and move to zoom in and out

Middle button click to center on atom

Right button hold and move to translate in the screen plane

Shift + middle button scroll to adjust clipping

Shift + left button hold and move to zoom in and out

Ctrl + left button hold and move to translate in the screen plane

Ctrl + right button hold and move to rotate in the screen plane