Name: Running and Analyzing MD Simulation to Study Complex Membrane Systems
Uploaded: 2023-09-01T14:10:00
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running and analyzing md simulation to study complex membrane systems

Molecular Dynamics를 사용한 Lipid Bilayers 시뮬레이션 및 분석

Lipids are major constituents of membranes, which provide boundaries for cells and enable intracellular compartmentalization1,2,3. Lipids are amphiphilic, with a polar head group and two hydrophobic fatty acid tails; these self-assemble into a bilayer to minimize contact of the hydrophobic chains with water3,4. Various combinations of hydrophilic head groups and hydrophobic tails result in different classes of lipids in biological membranes, such as glycerophospholipids, sphingolipids, and sterols (Figure 1)1,5,6. Glycerophospholipids are primary building blocks of eukaryotic cell membranes composed of glycerophosphate, long-chain fatty acids, and head groups of low-molecular weight7. Lipid nomenclature is based on differences in head groups; examples include phosphatidyl-choline (PC), phosphatidyl-ethanolamine (PE), phosphatidyl-serine (PS), phosphatidyl-glycerol (PG), phosphatidyl-inositol (PI), or the unmodified phosphatidic acid (PA)5,6. As for hydrophobic tails, the length and degree of saturation vary, along with the backbone structure. The possible combinations are numerous, resulting in thousands of lipid species in mammalian cells6. Changes in membrane lipid composition lead to different mechanical and structural membrane properties that impact the activity of both integral membrane proteins and peripheral proteins2,6.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65712/65712fig01.jpg" /> 
Figure 1. Representative lipid structures. Fatty acid tails are shown in blue boxes, common lipid head groups in orange, and sample backbones in purple. <a href="https://www.jove.com/files/ftp_upload/65712/65712fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Lipids are active players in cellular processes, protein activation in signaling cascades, and healthy cell homeostasis8,9. Altered lipid dynamics are the result of infection or can be markers of pathogenesis of disease10,11,12,13,14,15. As barriers for the cell, the study of membrane lipids and their role in permeation of small molecules is of relevance for drug delivery systems and membrane disruption mechanisms16,17. Chemical diversity and different ratios of lipid species across organelles, tissues, and organisms give rise to complex membrane dynamics2. It is therefore important to retain these characteristics in modelling studies of lipid bilayers, especially when the goal of a study is to examine interactions of other biomolecules with the membrane. The lipid species to consider in a model depends on the organism and cellular compartment of interest. For example, PG lipids are important for electron transfer in photosynthetic bateria18, while phosphorylated inositol lipids (PIPs) are major players in plasma membrane (PM) dynamics and signaling cascades in mammalian cells19,20. Inside the cell, the PM, endoplasmic reticulum (ER), Golgi, and mitochondrial membranes contain unique lipid abundances that influence their function. For example, the ER is the hub for lipid biogenesis and transports cholesterol out to the PM and Golgi; it contains a high lipid diversity with an abundance of PC and PE, but low sterol content, which promotes membrane fluidity21,22,23,24. In contrast, the PM incorporates hundreds and even thousands of lipid species depending on the organism25, it contains high levels of sphingolipids and cholesterol that give it a characteristic rigidity compared to other membranes in the cell24. Leaflet asymmetry should be considered for membranes like the PM, which has an outer leaflet rich in sphingomyelin, PC, and cholesterol, and an inner leaflet rich in PE, PI and PS that are important for signaling cascades24. Finally, lipid diversity also prompts the formation of micro-domains that differ in packing and internal order, known as lipid rafts24,26; these exhibit lateral asymmetry, are hypothesized to play important roles in cellular signaling26, and are hard to study due to their transient nature.
Experimental techniques such as fluoroscopy, spectroscopy, and model membrane systems like giant unilamellar vesicles (GUVs) have been used to investigate interactions of biomolecules with membranes. However, the complex and dynamic nature of the components involved is difficult to capture with experimental methods alone. For example, there are limitations on the imaging of transmembrane domains of proteins, the complexity of membranes used in such studies, and the identification of intermediate or transient states during the process of interest27,28,29. Since the advent of molecular simulation of lipid monolayers and bilayers in the 1980s29, lipid-protein systems and their interactions can now be quantified at the molecular level. Molecular dynamics (MD) simulation is a common computational technique that predicts the movement of particles based on their intermolecular forces. An additive interaction potential describes the bonded and non-bonded interactions between particles of the system30. The set of parameters used to model these interactions is termed the simulation forcefield (FF). These parameters are obtained from ab initio computations, semi-empirical, and quantum mechanical calculations, and optimized to reproduced data from X-ray and electron diffraction experiments, NMR, infrared, Raman and neutron spectroscopy, amongst other methods31.
MD simulations can be used to study systems at various levels of resolution32,33,34. Systems that aim to characterize specific biomolecular interactions, hydrogen bonds, and other high-resolution details are studied with all-atom (AA) simulations. In contrast, coarse-grained (CG) simulations lump atoms into larger functional groups to reduce computational cost and examine larger scale dynamics33. Situated in between these two are united-atom (UA) simulations, where hydrogen atoms are combined with their respective heavy atoms to accelerate the computation33,35. MD simulations are a powerful tool for exploration of the dynamics of lipid membranes and their interactions with other molecules and can serve to provide molecular level mechanisms for processes of interest at the membrane interface. Additionally, MD simulations can serve to narrow down experimental targets and predict macromolecular properties of a given system based on microscopic interactions.
In brief, given a set of initial coordinates, velocities, and a set of conditions like constant temperature and pressure, positions and velocities of each particle are calculated through numerical integration of the interaction potential and Newton&#39;s Law of motion. This is repeated iteratively, thereby generating a simulation trajectory30. These calculations are performed with an MD engine; among several open-source packages, GROMACS36 is one of the most commonly used engines and the one we describe here. It also includes tools for analysis and constructing initial coordinates of systems to be simulated37. Other MD engines include NAMD38; CHARMM39, and AMBER40, which the user can select at their own discretion based on computational performance of a given system. It is critical to visualize the trajectories during the simulation as well as for analysis and interpretation of the results. A variety of tools are available; here we discuss visual molecular dynamics (VMD) that offers a wide range of features, including three dimensional (3-D) visualization with expansive drawing and coloring methods, volumetric data visualization, building, preparing, and analyzing trajectories of MD simulation systems, and trajectory-movie making with no limits on system size, if the memory is available41,42,43.
The accuracy of predicted dynamics between system components is directly influenced by the FF chosen for the propagation of the trajectory. Empirical FF parametrization efforts are pursued by few research groups. The most established and common FF for MD include CHARMM39, AMBER40, Martini44, OPLS45, and SIRAH46. The all-atom additive CHARMM36 (C36) force field47 is widely used for AA MD of membrane systems as it accurately reproduces experimental structural data. It was originally developed by the CHARMM community, and it is compatible with multiple MD engines like GROMACS and NAMD. Despite improvements across common FFs, there is a continuous effort to improve the parameter sets to allow predictions that closely reproduce experimental observables, driven by interests in particular systems of study48,49.
A challenge when simulating lipid membranes is determining the length of simulation trajectory. This is largely dependent on the metrics to be analyzed and the process that one aims to characterize. Typically, complex lipid mixtures require longer time to reach equilibrium, as more species must have enough time to diffuse on the membrane plane and reach a stable lateral organization. A simulation is said to be at equilibrium when the property of interest has reached a plateau and fluctuates about a constant value. It is common practice to obtain at least 100-200 ns of equilibrated trajectory to carry out appropriate statistical analysis on the properties and interactions of interest. It is common to run membrane-only simulations between 200-500 ns, depending on the complexity of the lipid mixture and research question. Protein-lipid interactions typically require longer simulation times, between 500-2000 ns. Some approaches to accelerate sampling and observable dynamics with membrane systems are: (i) the highly mobile membrane mimetic (HMMM) model, which replaces end carbons of lipids in the membrane with organic solvent to accelerate sampling50; and (ii) hydrogen mass repartitioning (HMR), which combines a fraction of the masses of heavy atoms within a system with those of hydrogen atoms to allow the use of a larger simulation timestep51.
The following protocol discusses a beginner-friendly approach to build, run, and analyze realistic membrane models using AA MD. Given the nature of MD simulations, multiple trajectories must be run to account for reproducibility and proper statistical analysis of the results. It is current practice to run at minimum three replicas per system of interest. Once the lipid species have been selected for the organism and process of interest, basic steps to build, run, and analyze a simulation trajectory of a membrane-only system are outlined and summarized in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65712/65712fig02.jpg" /> 
Figure 2. Schematic to run MD simulations. Orange boxes correspond to the three main steps described in the protocol. Underneath is the workflow of the simulation process. During system setup, the system containing the initial coordinates of a solvated membrane system is built with a system input generator like CHARMM-GUI Membrane Builder. After transferring the input files to a high-performance computing cluster, the simulation trajectory is propagated using an MD engine, such as GROMACS. Trajectory analysis can be done on the computer cluster or a local working station along with visualization. Analysis is then carried out, using either packages with built-in analysis code such as GROMACS and VMD, or by using Bash scripts or various Python libraries. <a href="https://www.jove.com/files/ftp_upload/65712/65712fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

저자 스포트라이트: Advancing Cell Membrane Biophysics - Exploring Interactions and Challenges Through Experimental and Computational Approaches(세포막 생물물리학의 발전 - 실험 및 계산적 접근법을 통한 상호 작용 및 과제 탐색) 

시뮬레이션 연구에서 복잡한 지질 혼합물을 사용한 사실적인 막 모델링

Cell membrane biophysics is rapidly growing. Common experimental approaches include fluorescence microscopy, neutron and X-ray scattering, mass spectroscopy, and atomic force microscopy. Computational approaches include phonological models and those based on statistical thermodynamics that help us understand atomistic or molecular level details of interactions occurring at the membrane interface and inside its hydrophobic core.When using molecular dynamic simulations, challenges include sampling events of interest, setting a simulations length, ensuring results converge and reproduce physical values, and accessing computational power. These challenges are particularly true for transient events at the membrane interface, such as the interaction of peripheral proteins with it or the aggregation of lipids and proteins that require large changes in confirmation. This protocol provides a beginner friendly step-by-step guide to start running molecular dynamic simulations of complex lipid membranes.There are many software alternatives to carry these simulations, and some packages have tutorials or manuals. We hope our protocol provides a concise foundation on realistic membrane modeling and tips on the considerations that influence the quality of results and these types of modeling studies. This protocol has allowed us to capture interactions between membrane lipids and other biomolecules that were not observed using pure or binary lipid mixtures.Many interactions at the membrane surface depend on the diversity of lipids on the membrane itself. Our models demonstrate the importance of incorporating appropriate lipid species to accurately explore biomolecular function within membranes.

Watch this Scientific Journal Video about Running and Analyzing MD Simulation to Study Complex Membrane Systems at JoVE.com

Running and Analyzing MD Simulation to Study Complex Membrane Systems  

복잡한 멤브레인 시스템을 연구하기 위한 MD 시뮬레이션 실행 및 분석

분자 역학 시뮬레이션을 위한 시스템 좌표를 작성한 후 작성된 좌표계를 최소화하고 완화하는 제출 스크립트를 작성합니다. CHARMM-GUI에서 제출 스크립트로 출력에 제공된 README 파일의 해시태그 생성 주석까지 나열된 명령을 사용합니다. 완화 스크립트를 제출하고 프로덕션 실행을 진행하기 전에 해당 단계의 모든 출력 파일이 생성되었는지 확인합니다.완료되면 6단계 완화 실행 중에 GROMACS에서 생성된 다른 확장자를 가진 출력 파일이 있는지 확인합니다. 벤치마킹을 수행하려면 1-2나노초 길이의 짧은 궤적을 생성하고 다양한 수의 컴퓨팅 노드를 사용하여 계산 비용을 추정합니다. 서로 다른 수의 컴퓨팅 노드에 대한 성능을 하루에 나노초 단위로 비교하여 실행에 가장 적합한 리소스를 결정합니다.최대 성능의 75%에서 80% 사이의 성능 수준이 되는 노드 수를 선택합니다. 파일 형식을 xtc 파일로 변경하거나 프레임 크기를 줄여 GROMACS에서 trr 파일로 표시된 원시 궤적 파일을 압축하여 시각화 및 분석을 위해 로컬 스테이션으로 효율적으로 전송할 수 있습니다. 분석을 실행하기 전에 전체 궤적을 시각화하여 관심 분자 또는 원자를 식별하고 특성 분석을 위한 궤적 부분을 결정합니다.그런 다음 멤브레인 전용 시뮬레이션을 위한 지질 시계열당 면적을 결정하고 궤적의 일부를 식별합니다. 막 구조를 분석한 결과, 소포체에 대한 두 모델 간에 두께에 상당한 차이가 있는 것으로 나타났으며, 이는 지질당 면적과 막 두께 사이의 반비례 관계를 나타냅니다. 각 지질 종의 중수소 순서 매개변수는 PI 모델에서 SN1 꼬리에 대해 약간의 증가를 보여주는 DPPE를 제외하고 모델 간의 지질 꼬리 순서 간에 차이가 거의 또는 전혀 없음을 보여주었습니다.지질 조성 및 막 모델은 다른 분자와의 상호 작용을 조절합니다. 예를 들어, PC와 PS 지질의 서로 다른 비율을 사용한 시뮬레이션은 정전기 상호 작용이 음이온 지질에 대한 비국소화된 친유성 양이온인 D112의 초기 결합을 유도하고 소수성 상호 작용이 분자를 막 코어로 끌어당긴다는 것을 보여주었습니다.

running-and-analyzing-md-simulation-to-study-complex-membrane-systems

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Running and Analyzing MD Simulation to Study Complex Membrane Systems

Realistic Membrane Modeling Using Complex Lipid Mixtures in Simulation Studies

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different mechanical and structural properties in the membrane that directly impact the molecules and processes that occur at this interface. Lipid composition is dynamic and can serve to modulate cell signaling processes. Computational approaches are increasingly used to predict interactions between biomolecules and provide molecular insights to experimental observables. Molecular dynamics (MD) is a technique based on statistical mechanics that predicts the movement of atoms based on the forces that act on them. MD simulations can be used to characterize the interaction of biomolecules. Here, we briefly introduce the technique, outline practical steps for beginners who are interested in simulating lipid bilayers, demonstrate the protocol with beginner-friendly software, and discuss alternatives, challenges, and important considerations of the process. Particularly, we emphasize the relevance of using complex lipid mixtures to model a cell membrane of interest to capture the appropriate hydrophobic and mechanical environments in simulation. We also discuss some examples where membrane composition and properties modulate the interactions of bilayers with other biomolecules.

Membrane lipid diversity in structure and composition is an important contributor to cellular processes and can be a marker of disease. Molecular dynamics simulations allow us to study membranes and their interactions with biomolecules at atomistic resolution. Here, we provide a protocol to build, run, and analyze complex membrane systems.

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different ...

연구

생물학

Building the System Coordinates for Molecular Dynamics Simulations

To begin, use a web browser to visit the charmm-gui. org website. Create and activate your free account before building your first set of files.Go to the top menu, navigate to the input generator and select membrane builder from the available options on the left side of the screen. Select the bilayer builder to construct a lipid bilayer, then select the membrane only system and store the generated job ID for future access. Visualize the systems during every step of the building process by clicking view structure located at the top of the page.Consistently inspect for missing components or errors in patch size. Select the heterogeneous lipid option regardless of whether to build a single component bilayer, then choose a rectangular box type. For hydration, select 45 water molecules per lipid, which is ample for a fully hydrated bilayer.Now set the length of XY based on the number of lipid components. Determine the number of lipids needed for each lipid species based on the pre-planned model. To represent the endoplasmic reticulum and eukaryotic cells, a combination of 336 DOPC, 132 DPPE, 60 cholesterol, and 72 POPI lipids was used in the PI model while 330 DOPC, 126 DPPE, 54 cholesterol, 66 POPI, and 24 DOPS lipids were used for the PIPS model.Input the desired number of molecules for the upper and lower leaflets in the two boxes next to the lipid name to create a symmetric membrane composition. Then navigate to the top of the lipid species list and initiate the action by clicking on the show the system info button. Using CHARMM-GUI, validate the total count of lipids in each leaflet in the symmetric bilayer.Continue clicking the next screens to set the desired values for temperature and pressure for simulation and the syntax for the simulation files according to the molecular dynamics engine of choice. For instance, GROMACS. Download the resulting files and transfer them to the computer cluster.Use selected software, for instance, visual molecular dynamics or PyMOL to visualize the final system.

분자 역학 시뮬레이션을 위한 시스템 좌표 구축

After building the system coordinates for molecular dynamics simulations, create a submission script to minimize and relax the built coordinate system. Use the commands listed until hashtag production comment in the README file provided in the output from CHARMM-GUI to the submission script. Submit the relaxation script and ensure all output files from its steps have been produced before progressing to the production run.Upon completion, verify the presence of output files with different extensions generated from GROMACS during the six-step relaxation run. To perform benchmarking, generate short trajectories one to two nanoseconds long and estimate the computational cost using varying numbers of computing nodes. Compare the performance in nanoseconds per day for different numbers of computing nodes to determine the optimal resources for the run.Choose the number of nodes that result in a performance level between 75 to 80%of the maximum. Compress raw trajectory files denoted as trr files in GROMACS by changing the file format to xtc files or skipping frames to reduce file size to facilitate efficient transfer to the local station for visualization and analysis. Visualize the full trajectory before running any analysis to identify the molecules or atoms of interest and determine the trajectory portion intended for characterization.Then determine the area per lipid time series for membrane only simulations and identify the portion of the trajectory. Analysis of membrane structure revealed a significant difference in thickness between the two models for the endoplasmic reticulum, indicating an inverse relationship between the area per lipid and membrane thickness. Deuterium order parameters of each lipid species showed that there is little to no difference between the order of lipid tails between the models except for DPPE, which shows a slight increase for the SN1 tail in the PI model.Lipid composition and membrane models modulates interactions with other molecules. For example, simulations using different ratios of PC and PS lipids showed that electrostatic interactions drive the initial binding of D112, a delocalized lipophilic cation to anionic lipids and hydrophobic interactions pull the molecule into the membrane core.