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Sep 2019

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Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
用NAMD和PTools/Heligeom模拟蛋白质丝的微尺度和中尺度扰动   

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Abstract

Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are difficult to model due to the helical character of the system. Here, we describe how distortions in RecA nucleofilaments and their consequences on the filament-DNA and bound DNA-DNA interactions at different stages of the homologous recombination process can be modeled using the PTools/Heligeom software and subsequent molecular dynamics simulation with NAMD. Modeling methods dealing with helical macromolecular objects typically rely on symmetric assemblies and take advantage of known symmetry descriptors. Other methods dealing with single objects, such as MMTK or VMD, do not integrate the specificities of regular assemblies. By basing the model building on binding geometries at the protomer-protomer level, PTools/Heligeom frees the building process from a priori knowledge of the system topology and enables irregular architectures and symmetry disruption to be accounted for.


Graphical abstract:



Model of ATP hydrolysis-induced distortions in the recombinant nucleoprotein, obtained by combining RecA-DNA and two RecA-RecA binding geometries.


Keywords: Helical filament (螺旋丝), Mesostructure (细观结构), Homologous recombination (同源重组), Integrative modeling (综合建模), Molecular dynamics simulation (分子动力学模拟), Nucleic acid (核酸)

Background

Oligomeric filamentous assemblies, made of the quasi-regular repetition of macromolecular protomers, are essential components of the cell. Filaments from the cytoskeleton support cell walls or form intracellular transport networks. Other types of filaments are involved in the maintenance, segregation, and repair of DNA (Ghosal and Löwe, 2015). These processes involve dynamic interactions between two generalized helices, the DNA helix and the filament helix, generally accompanied by changes in the DNA linking number and sometimes with coordinated changes in the filament topology. Elucidation of the structure of protein filaments using structural biology methods such as X-ray crystallography or Nuclear Magnetic Resonance has long been highly challenging due to their large size and difficulty in forming crystals. With the recent advances in Cryo-Electron Microscopy, many filament structures are being unveiled; however, filament plasticity and dynamic events are still difficult to capture, and model building remains necessary to decipher how filaments respond to external stimuli. Methods that have been proposed for the modeling of filaments are mostly based on specific docking approaches (Inbar et al., 2005; Casciari et al., 2006; Karaca et al., 2010; Esquivel-Rodriguez et al., 2012) and generally take advantage of known symmetry characteristics (Eisenstein et al., 1997; Berchanski et al., 2003 and 2005; Comeau and Camacho, 2005; Pierce et al., 2005; Schneidman-Duhovny et al., 2005). These methods are not appropriate for investigating structural perturbations in the filament, either global (changes in helical characteristics) or local (symmetry disruption), that appear as responses to external stimuli.


The Heligeom module (Boyer et al., 2015) of the PTools library software (Saladin et al., 2009) has been developed to meet the challenge of modeling helical protein filaments, their interactions with other molecules such as DNA, and their structural responses to external stimuli, either diffuse (changes in longitudinal or torsional stress, following changes in salt concentration or pH), or focused (local molecule binding or interface modifications). Heligeom derives the geometry of oligomeric assemblies from the binding geometry between pairs of neighboring protomers, which offers the possibility to combine several binding modes within the same filament. The PTools library itself gathers a collection of tools to manipulate macromolecules or selected regions of these macromolecules in atomic or coarse-grained representation. While it shares several functionalities with other libraries such as MMTK (Hinsen, 2000) or the VMD Script Library (Humphrey et al., 1996), certain functionalities such as the easy use of screw transformations make it particularly well suited to dealing with helical or quasi-helical assemblies.


Here, we present the application of PTools/Heligeom to model an irregular form of the RecA nucleofilament complex that is active in homologous recombination and includes one to three DNA strands (Boyer et al., 2015). Distortion in the filament results from the introduction of an alternative binding geometry of RecA-RecA interaction at the center of a two-turn helical nucleofilament (twelve monomers). Whereas the principal RecA-RecA binding mode corresponds to the favorable binding geometry between two RecA monomers in the presence of ATP, the alternative binding mode corresponds to the geometry in the presence of ADP. Both binding geometries have been solved by crystallography. The model therefore represents the result of one ATP molecule being hydrolyzed in the filament center. All three DNA strands participate in a strand exchange process. Strand 1 is a damaged DNA strand on which the filament has polymerized; it is bound to DNA-binding site I in the filament. Strand 3 is homologous to strand 1 and occupies the secondary binding site (site II). Strand 2 is complementary to both strand 1 and strand 3. During the strand exchange process catalyzed by the filament, strand 1 captures strand 2, which was initially paired with strand 1. We model three states of the system: the initial state, where only strand 1 is bound to site I (one bound strand); the annealed state, where strand 2 has paired with strand 1 at site I (two bound strands); and the state resulting from strand exchange, where strands 1 and 2 are at site I and strand 3 is at site II (three bound strands). The protocol presented here can be used for any oligomeric system in which three-dimensional structures are available for at least two protein-protein binding geometries obtained under two different conditions, for example, with different cofactors, and present regions of structural similarity. The protocol enables exploration of transient intermediate geometries that are difficult to access experimentally. It is particularly well suited to the study of "collaborative protein filaments," as described in Ghosal and Lowe (2015).

Equipment

  1. Linux workstation (Dell) running a Linux distribution Ubuntu 18.04.5 LTS (GNU/Linux 4.15.0-118-generic x86_64)

  2. Access to a scalar supercomputer (petaflop performance) for molecular dynamics simulations

Software

  1. PTools (Saladin et al., 2009) is a python/C++ library dedicated to the manipulation, assembly, and analysis of macromolecular complexes. It is freely available in the GitHub repository at the address https://github.com/ptools/ptools/tree/develop; it is licensed under the GNU General Public License v3.0. The Heligeom module relates protomer-protomer binding geometries to the geometry of large oligomeric assemblies (Boyer et al., 2015). Heligeom basic functions consist of analyzing protomer interfaces in terms of the helical parameters (Figure 1A) of associated screw objects – pitch (P), number of monomers per turn (N), handedness, see Figure 1A, right – and constructing helices with the desired number of protomers. The helix can optionally be aligned to the Z axis at the origin. Figure 1B shows an example of the workflow of PTools/Heligeom functions that are commonly used in this work. These include selection functions (chains, residue numbers or types, atoms numbers or types), the superposition function that outputs a transformation matrix, and the mat_trans_to_screw function that produces the screw object corresponding to the transformation matrix.

  2. The NAMD software (Scalable Molecular Dynamics, Mackerell et al., 2004; Phillips et al., 2005; http://www.ks.uiuc.edu/Research/namd/) is used to perform energy minimization and molecular dynamics (MD) simulations.

  3. VMD (Humphrey et al., 1996) is a molecular visualization program: VMD can display and analyze large biomolecular systems and their MD trajectories using 3-D graphics and built-in scripting https://www.ks.uiuc.edu/Research/vmd/script_library/.

    NAMD and VMD are both developed at the NIH Center for Macromolecular Modeling & Bioinformatics, University of Illinois at Urbana-Champaign, by the Theoretical and Computational Biophysics Group. Both are distributed free of charge with source code.



    Figure 1. PTools/Heligeom. (A) Left: scheme of a screw transformation between two proteins A and B, with structurally similar core regions coreA and core2; right: calculation of the number of monomers per turn (nb), the pitch and the handedness (R: right, L: left) from the screw parameters θ and trans. (B) Typical PTools operations that enable the combination of available binding geometries. This includes creating Rigidbody objects from the .pdb files ("import .pdb files…"), defining structurally superposable regions with identical sizes using the select_resid_range attribute ("select a protein region"), defining mat, the transformation matrix that superposes the defined regions using the superpose function ("superpose selected regions…"), and defining the screw transformation hp equivalent to mat; example of how to use the screw object hp to create a new Rigidbody object according to the desired screw transformation is given under "apply transformation matrix…"

Procedure

  1. Filament construction

    1. Preparing building blocks

      1. Binding geometries are defined by pairs of interacting RecA monomers extracted from the RecA crystal structures with PDB codes 2REB (RecA bound to ADP molecules) and 3CMW (RecA bound to ATP and two DNA strands at site I). From each of these structure files, manually extract two neighboring monomers to define the corresponding binding geometry (monomers with different binding geometries are schematized in Figure 2A). In the case of 3CMW, in which binding geometries slightly differ along the 5-monomer filament, select central monomers (position 3 and 4) to avoid possible end effects. Make sure that the two monomers have the same number of residues and are structurally close (RMSD on CA atoms < 3 Å); otherwise, select structurally similar regions using the commands given in reference (Saladin et al., 2009) and Figure 1B. The pair of monomers extracted from the 2REB structure will be denoted RA and RB, and those extracted from 3CMW, CA and CB (Figure 2A). The corresponding new .pdb files should be named RA.pdb, RB.pdb, CA.pdb, CB.pdb (see Note 1).

      2. Building blocks are formed by monomer CA (resp. CB) with bound ATP/Mg2+ together with one, two, or three bound DNA segments. To define the DNA segments that are part of the building blocks, proceed as follows: for each of the two strands at site I, extract the three nucleotides that are bound to CA (resp. CB) in the 3CMW structure (the stoichiometry of RecA/DNA association is 3 nucleotides/monomer); note that determination of the residue numbers of the target nucleotides may require visual inspection of the 3CMW structure using VMD. The strand at site II is taken from our model published in Yang et al. (2015) (.pdb file is available upon request): select one RecA monomer and the bound DNA. Then, using PTools commands (Figure 1B), superpose the RecA monomer onto CA (resp. CB), calculate the transformation matrix associated with the superposition, and apply this transformation matrix to the DNA segment. Finally, for each number of strands, generate a .pdb file, named CA_x.pdb where x = 1, 2, or 3, that contains the CA monomer and its associated ATP molecule, magnesium ion, and DNA segment(s).

    1. Assembling building blocks

      1. Construct a 6-monomer filament segment with geometry characteristic to 3CMW using PTools/Heligeom (see Figure 2B) via the command line:

        $ ptools heligeom -n 6 -o BLOCK1.pdb -Z CA_x.pdb CB_x.pdb

      2. In a python script into which the PTools package has been imported, construct the Rigidbody objects corresponding to RA, RB, and BLOCK1 (resp. RA, RB, and BL1). Select chain A of BL1 and store it in a Rigidbody object B1. Select chain F of BL1 and store it in a Rigidbody object B6. Superpose RA onto RB and output the corresponding transformation matrix mat1 (Figure 2A). Apply mat1 onto B6, thereby generating a monomer B7 that adjoins B6 following the binding geometry characteristic to 2REB (Figure 2C). Superpose B1 onto B7 and calculate the transformation matrix mat2 (Figure 2D); apply mat2 onto BL1, thus generating a new 6-monomer filament segment BL2 adjacent to BL1, with the binding geometry between the last unit of BL1 and the first unit of BL2 being 2REB type (Figure 2E and 2F).



    Figure 2. Construction of a composite filament. (A) Schematic representation of the building blocks that define two RecA-RecA binding modes: (top) the CA-CB type and (bottom) the RA-RB type; calculation of the transformation matrix mat1 that relates Rigidbody objects RA and RB (arrow). (B) Applying the heligeom command line to building blocks CA and CB produces a 6-unit helix with interfaces of the CA-CB type (Step A2a). (C) Generation of Rigidbody B7 from B6 by applying mat1; B6 and B7 interact via a RA-RB interface. (D) Calculation of mat2 that transforms B1 into B7. (E) Applying mat2 to the BL1 block. (F) Final result (see text in A.2.b). PTools object names are written in blue when used and in red when defined. The process is illustrated in the accompanying video at Figure2_video.mp4.


    1. Refining the junction between building blocks

      1. Adjust the interface flexible groups by replacing the N-terminal domain (one helix and associated linker, residues 1 to 37) of Rigidbody B7 with the equivalent region from RA. To this end, create an empty Rigidbody object BL2_adjusted. Select the Cα atoms of residue numbers 38 to 156, 165 to 193, and 213 to 333 of RA (non-flexible regions) and store them in the Rigidbody SRA. Perform the same selection for B7, stored in SB7; superpose SRA onto SB7 and output the transformation matrix mat3. Select the N-terminal domain of RA, residues 1 to 37, and store the selection in NT; store residues 38 to 333 of B7 in CB7; store chains B, C, D, E, and F of BL2 in BL2_BtoF. Apply mat3 to NT and store the result in NT7. Successively concatenate NT7, CB7, and BL2_BtoF to BL2_adjusted; concatenate BL2_adjusted to BL1. Output the structure in PDB format, named FINAL_x.pdb, where x = 1, 2, 3 is the number of DNA strands. Three final structures have now been generated.

      2. Process the final structures in such a way as to group the DNA fragments, label them with a specific chain name, group the ATP molecules and change their chain name, and name the protein chains from A to L (Figure 2C, right); replace the ATP/Mg2+ bound to chain F with ADP. This can be done within the python/PTools script before outputting the .pdb files in the previous paragraph or manually by processing the .pdb files using a text editor.

    2. Closing DNA chains with restrained minimization for each file FINAL_x.pdb, x = 1, 2, 3

      1. Using Recipe 1, roughly reposition the trinucleotide DNA segment T3F from strand 3 associated with monomer F. This segment presents numerous steric clashes with monomer G following the change in filament axis orientation, consecutive to the modification of the F-G interface geometry. The change in orientation also produces an important rupture of site II continuity between T3F and the DNA segment T3G of strand 3 associated with monomer G.

      2. Generate a topology .psf file using the «Automatic PSF builder» VMD extension.

      3. Create a constraint file from the FINAL_x.pdb file for NAMD minimization by adding «1.00» to column 62 of the restrained atoms (P for the DNA chains and CA for proteins); the script given as Recipe 2 below can be used to this end. The resulting file FINAL_x_cstr.pdb needs to be modified in order to free the phosphate atoms situated at the junction between chains F and G for energy minimization, where the helix symmetry has been broken, by replacing «1.00» with «0.00» for these specific phosphate atoms (three phosphate atoms are freed per DNA chain).

      4. Perform 5000 steps of conjugate gradient energy minimization using NAMD2.10. This enables closure of the DNA backbones by restoring the distances between covalently linked atoms according to the topology file and releasing small steric clashes if any.


  2. Stability and evolution of the filament model: molecular dynamics simulations

    1. Simulation preparation for each file FINAL_x.pdb, x = 1, 2, 3

      1. Immerse the structure in a water box using the VMD extension «Add Solvation Box»; use a TIP3P water model and a box padding value of 10 Â.

      2. Add Na+ and Cl- ions to the water box using the VMD extension «Add Ions»; select the option «Neutralize and set NaCl concentration to 0.15 mol/L».

    2. Simulation runs for each of the three systems using NAMD2.10

      Given the large size of the systems (about 450,000 atoms), this step needs to be performed on distributed computer nodes, for example, in a computer center. The simulation follows standard NAMD protocols.

      1. Simulation conditions: use the CHARMM 27 force field including CMAP corrections; use periodic boundary conditions, with the particle mess Ewald method to account for long range interactions and a smooth switch of van der Waals interactions between 10 and 12 Å; set time steps to 2 fs using the SHAKE algorithm; control the temperature and pressure using a Langevin dynamics scheme and a Nose-Hoover Langevin piston.

      2. Simulation run: perform 5000 steps of conjugate gradient minimization followed by a progressive heating stage up to 300K, a long equilibration phase of 30 ns, and a 100 ns production phase. During heating and equilibration, set restraints on the P and CA atoms of the files, with the force constant decreasing from 0.5 to 0.05 kcal·mol-1·Å-2. Do not use restraints during the production phase. Perform three independent simulations for the three-stranded system using identical conditions.

Data analysis

Results of the following analysis steps can be found in the Supplemental Information (SI) of Boyer et al. (2015).

  1. Conventional MD analysis. Check the stability of the energy and temperature during the molecular dynamics simulation trajectories using VMD tools. Reduce the size of the trajectory files by eliminating the water molecules. Plot the time evolution of root-mean-square fluctuation (RMSF) or deviation (RMSD) values of the system or selected regions of the system, taken on the Cα atoms: whole filament, filament center, DNA center (Boyer et al., 2015, Figure SI-7). To separate the fluctuations of each monomer from those of the entire filament, calculate the RMSD and RMSF values along the trajectory for each individual RecA monomer, after superposition restricted to this monomer; plot the evolution of the average value of the resulting RMSDs, taken on all monomers (Boyer et al., 2015, Figure SI-7); individually plot the RMSF corresponding to each monomer (Boyer et al., 2015, Figure SI-5). Visually inspect the final structures of trajectories using VMD to select atoms or groups of atoms of interest that modify their contacts with other groups as a result of the interface modification; plot the time evolution of these distances; calculate the corresponding distances in regular filaments and incorporate this value into the plot as a horizontal line as a reference (Boyer et al., 2015, Figure SI-3).

  2. Specific MD analysis for a filamentous assembly. Calculate the width of the groove entrance at the level of each monomer, as described in Boyer et al. (2015) (Materials and Methods); plot the time evolution of the width value for each monomer and then plot the value measured in regular filaments as in Boyer et al. (2015), Figure SI-2. Using PTools utilities, calculate the evolution, along the trajectory, of the fNAT values for each non-terminal interface, where fNAT is the fraction of pair contacts from the starting structure that are conserved in a given snapshot (a contact being defined as a pair of residues from each interacting monomer that are distant by less than 5 Å, and pair contacts being restricted to the non-flexible regions of the interface, as defined above in Step A3a) (Boyer et al., 2015, Figure SI-6).

  3. DNA analysis. Evolution of the interaction between DNA strands is monitored in two ways: by measuring the shortest phosphate-phosphate inter-strand distances between each pair of strands for each snapshot (see Boyer et al., 2015, Figure SI-9 for details) or by calculating the time evolution of pairing distances between three base pairs, selected by visual inspection using VMD (Boyer et al., 2015, Figure 4).

Notes

  1. In the 3CMW .pdb file, the residues are numbered from 1 to 333 for the first monomer, from 1001 to 1333 for the second, from 2001 to 2333 for the third, etc. It is advisable to edit the files CA.pdb and CB.pdb in order to have their residue numbering within the range of 1-333. Care must be taken that all modifications of .pdb files respect the .pdb file format.

  2. The models generated in this work were deposited into ModelArchive https://www.modelarchive.org/ with codes ma-900pk, ma-l1kfl and ma-eaaa9, and are publicly available at the following URLs: https://www.modelarchive.org/doi/10.5452/[code] (where [code] is either ma-900pk, ma-l1kfl or ma-eaaa9).

Recipes

  1. The T3F trinucleotide segment defined in Step A4a can be repositioned by following the commands below, where the chain index for strand 3 is "P," the T3F nucleotides are numbered from 16 to 18 in the 5’-3’ direction, nucleotide number 15 is associated with monomer E, and nucleotide 19 is the first nucleotide of T3G in the 5’ direction. The backbone discontinuity occurs between nucleotides 18 and 19. The script below defines three points, O, A, and B, where O is positioned on atom P of nucleotide 15, A on atom O3' of nucleotide 19, and B on atom P of nucleotide 18. T3F will be rotated by 40° around the axis (O,u), where u is the normalized cross product between vectors OA and OB:


    $ python

    >>> from ptools import *

    >>> fila = Rigidbody("FINAL_3.pdb")

    >>> strP = fila.select_chain_id("P").create_rigid()

    >>> t3f = strP.select_res_range(15,18).create_rigid()

    >>> atom=(strP.select_res_range(15,15) & \

    strP.select_atom_type("P")).create_rigid()

    >>> O = atom.get_coords(0)

    >>> atom=(strP.select_res_range(19,19) & \

    strP.select_atom_type("O3'")).create_rigid()

    >>> A = atom.get_coords(0)

    >>> atom=(strP.select_res_range(18,18) & \

    strP.select_atom_type("P")).create_rigid()

    >>> B = atom.get_coords(0)

    >>> OA = A - O

    >>> OB = B - O

    >>> u = Coord3D()

    >>> u.x = OA.y * OB.z - OA.z * OB.y

    >>> u.y = OA.z * OB.x - OA.x * OB.z

    >>> u.z = OA.x * OB.z - OA.z * OB.x

    >>> u.normalize()

    >>> newt3f = Rigidbody(t3f)

    >>> newt3f.ab_rotate(O,O+u,40*3.14/180.)

    >>> write_pdb(newt3f,"new_T3F.pdb")


    The file FINAL_3.pdb can then be edited to incorporate new_T3F.pdb in place of T3F.

  2. The following Linux command can be used to generate a constraint file for NAMD in order to restrain the displacement of P and CA atoms during energy minimization, heating, and the beginning of equilibration (see Step A4c):


    $ awk '$3 == "P" || $3 == "CA" { \

    > printf "%s 1.00%s\n",substr($0,1,61), substr($0,67)}; \

    > $3 != "P" && $3 != "CA" ' FINAL_x.pdb > FINAL_x_cstr.pdb


    This command labels all "P" and "CA" atoms of the file FINAL_x.pdb. In order for the minimization process to be able to close the DNA backbones, phosphate atoms from the junction regions must be released by replacing the "1.00" in columns 63 to 66 with "0.00". For example, for strand 3, if one keeps the notations from Recipe 1, phosphates 15 to 20 of chain "P" will be released.

Acknowledgments

The authors wish to acknowledge the ‘Initiative d’Excellence’ program of the French State for funding [DYNAMO, ANR-11-LABX-0011-01]. The original research paper from which this protocol is derived was published by Boyer et al. (2019). We thank Raquel de Miranda for assistance in preparing the video describing RecA filament preparation.

Competing interests

The authors certify that they have no competing interests.

Ethics

The protocol presented here did not use any human or animal subjects.

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简介

[摘要]蛋白丝是一种动态实体,通过轻微或大量改变连续原体之间的内部结合几何结构来响应外部刺激。这导致灯丝结构的整体变化,由于系统的螺旋特性,这很难建模。在这里,我们描述了如何在扭曲的RecA nucleofilaments及其对后果长丝-DNA和在同源重组过程的不同阶段结合的DNA-DNA相互作用可以被建模使用PTOOLS / Heligeom软件和随后的分子动力学模拟与NAMD。处理螺旋大分子的建模方法 cular 对象通常依赖于对称组件并利用已知的对称描述符。其他处理单个对象的方法,例如 MMTK 或 VMD ,没有集成常规程序集的特性。PTools / Heligeom将模型构建建立在原体-原体级别的结合几何结构基础上,将构建过程从系统拓扑的先验知识中解放出来,并能够解决不规则架构和对称破坏的问题。

图文人摘要:

在毒素重组ATP水解导致的扭曲现象的模型Ñ吨核蛋白,由得到的组合的RecA -DNA和两个RecA的-RecA的结合的几何结构。


[背景]由准规则重复的大分子原体组成的寡聚丝状组装体是细胞的重要组成部分。来自细胞骨架的细丝支撑细胞壁或形成细胞内运输网络。其它类型的长丝都参与了维护,偏析,和DNA(修复Ghosal和Löwe酒店,2015)。这些过程涉及两个广义螺旋(DNA 螺旋和细丝螺旋)之间的动态相互作用,通常伴随着 DNA 连接数的变化,有时伴随着细丝拓扑结构的协调变化。使用结构生物学方法如X射线晶体学或核磁共振蛋白丝的结构的阐明早已高度挑战由于其大尺寸和难度在形式荷兰国际集团的晶体。随着最近的进步在低温-电子显微镜,许多灯丝结构被推出; ^ h H但是,长丝塑性的ND动态事件仍难以捕捉,与模型建设仍然需要破译丝对外界刺激的反应。已经提出的方法对模型的ING细丝大多是基于特定的对接办法(因巴尔等,2005; Casciari等,2006; Karaca的等,2010; Esquivel的罗德里格斯等人。,2012)和通常利用已知的对称特性(Eisenstein等,1997;Berchanski等,2003 和 2005;Comeau和 Camacho,2005;Pierce等,2005;Schneidman-Duhovny等,2005)。这些方法不适合于长丝调查结构扰动,是全局的(变化小号螺旋特性)或局部(对称性破坏),即表现为对外界刺激的反应。

PTools库软件(Saladin等人,2009 年) 的Heligeom模块(Boyer等人,2015年)已开发用于应对对螺旋蛋白丝、它们与其他分子(如 DNA)的相互作用及其结构响应进行建模的挑战对外部刺激,无论是弥漫性(变化š在纵向或扭转应力,以下的变化盐浓度或pH),或聚焦(本地分子结合或接口的修改)。Heligeom从相邻原体对之间的结合几何结构推导出寡聚组件的几何结构,这提供了在同一细丝中组合多种结合模式的可能性。所述PTOOLS库本身集的工具操作大分子或这些大分子的选定区域中的原子或粗粒度表示的集合。而这股几个功能与其他库如MMTK(Hinsen ,2000)或VMD脚本库(汉弗莱等人,1996),某些功能,如容易使用螺钉变换使其特别绥泰德处理螺旋或准螺旋组件。

在这里,我们展示了PTools / Heligeom的应用,以模拟不规则形式的RecA核丝复合物,该复合物在同源重组中具有活性,包括一到三个 DNA 链(Boyer等人,2015 年)。由于在两圈螺旋核丝(十二个单体)的中心引入了RecA-RecA相互作用的替代结合几何结构,导致了丝的扭曲。主要的RecA-RecA结合模式对应于存在 ATP 时两个RecA单体之间有利的结合几何结构,而替代结合模式对应于存在 ADP 的几何结构。两种结合几何结构都已通过晶体学解决。因此,该模型代表了一个 ATP 分子在长丝中心水解的结果。所有三个 DNA 链都参与链交换过程。链 1 是受损的 DNA 链,细丝已在其上聚合;它与灯丝中的 DNA 结合位点 I 结合。链 3 与链 1 同源并占据二级结合位点(位点 II)。链 2 与链 1 和链 3 互补。在长丝催化的链交换过程中,链 1 捕获链 2,它最初与链 1 配对。我们模拟系统的三种状态:初始状态,其中只有链 1 与位点 I结合(单链结合);退火状态,其中链 2 与链 1在位点 I(两条结合链)配对;和链交换产生的状态,其中链 1 和 2位于位点 I,链 3位于位点 II(三个结合链)。本协议编这里可以用于任何低聚系统,其中三维结构可用于至少两种蛋白质-蛋白质结合所获得的几何形状下的两个不同的条件,例如,具有不同的辅因子,和结构相似的本地区。该协议能够探索难以通过实验访问的瞬态中间几何。它特别适合于“协同蛋白丝”的研究,如 ( Ghosal and Lo ̈ we , 2015) 中所述。

关键字:螺旋丝, 细观结构, 同源重组, 综合建模, 分子动力学模拟, 核酸



设备


1个Linux工作站(戴尔)运行一个大号inux下分发的Ubuntu 18.04.5 LTS(GNU / Linux的4.15.0-118泛型x86_64的)                        2使用标量超级计算机(petaflop 性能)进行分子动力学模拟                        软件


1个PTOOLS (萨拉丁等人,2009)是一个Python / C ++库专用于操纵,组件,和大分子复合物的分析。它可以在 GitHub 存储库中免费获得,地址为https://github.com/ptools/ptools/tree/develop;它在 GNU 通用公共许可证 v3.0 下获得许可。所述Heligeom模块涉及原体,原体结合几何形状以大低聚组件的几何形状(博耶等人,2015)。Heligeom基本功能包括的分析原体在螺旋参数(图1的方面接口甲关联的螺钉的对象)              –螺距 (P)、每圈单体数 (N)、旋向性,参见图 1A,右–并构建具有所需数量的原体的螺旋。螺旋线可以选择在原点与 Z 轴对齐。图1乙示出的示例的工作流PTOOLS / Heligeom常用于这项工作中使用的功能。这些包括选择功能(链,残基数目或类型,原子数目或类型的),其输出的变换矩阵的叠加功能,和mat_trans_to_screw产生螺杆对象对应功能荷兰国际集团的变换矩阵。


2 NAMD 软件(Scalable Molecular Dynamics,Mackerell等人,2004 年;Phillips等人,2005 年;http://www.ks.uiuc.edu/Research/namd/)用于执行能量最小化和分子动力学( MD)模拟。            3 VMD(Humphrey等人,1996 年)是一个分子可视化程序:VMD 可以使用 3-D 图形和内置脚本来显示和分析大型生物分子系统及其 MD 轨迹https://www.ks.uiuc.edu/研究/vmd/script_library/。            NAMD 和 VMD 均由 NIH 大分子建模与生物信息学中心、伊利诺伊大学厄巴纳-香槟分校的理论和计算生物物理学小组开发。两者都随源代码免费分发。


图 1. PTools/Heligeom。(A)大号EFT:两个蛋白质之间的螺纹变换方案小号甲和乙,用结构上相似的核心区域科雷亚和核2 ; 右图:根据螺杆参数 θ 和反式计算每转的单体数量 ( nb )、螺距和旋向性(R :右,L :左)。(B)Ť ypical PTOOLS OPERAT离子,使所述COMBIN的通货膨胀可用结合的几何形状。这包括创建刚体的对象。PDB文件(“进口。PDB文件...”),定义在结构上叠加的使用所述相同大小的区域select_resid_range属性(“选择的蛋白质区域”),定义垫,该转换矩阵,它叠加使用的限定的区域叠加功能(“叠加选定区域...") ,并定义与mat等效的螺旋变换hp ;在“应用变换矩阵...”下给出了如何使用螺旋对象hp根据所需的螺旋变换创建新的刚体对象的示例。


程序


一个灯丝建设                1准备积木                IA乙inding几何形状是通过对相互作用的定义RecA的从提取的单体的RecA晶体结构与PDB代码2REB(RecA的绑定到ADP分子)和3CMW(RecA的绑定到ATP和两条DNA链在站点I)。从这些结构文件中的每一个中,手动提取两个相邻的单体以定义相应的结合几何结构(具有不同结合几何结构的单体在图 2A 中进行了示意图)。在3CMW的情况下,在其中结合地理metries略微沿5不同-单体长丝,选择中央的单体(位置3和4),以避免可能的最终的效果。确保两个单体具有相同数量的残基并且结构接近(CA 原子上的 RMSD < 3 Å );否则,使用参考(Saladin等人,2009 年)和图 1B 中给出的命令选择结构相似的区域。从 2REB 结构中提取的单体对将表示为 R A和 R B ,而从 3CMW 、C A和 C B 中提取的单体将表示为(图 2A)。对应的新 . pdb文件应命名为RA.pdb 、RB.pdb 、CA.pdb 、CB.pdb (见注 1 )。          IB构建块是由单体C形成甲(分别ç乙与结合的ATP / Mg)的2+被一个,两个一起,或三个结合的DNA片段。要定义作为构建块一部分的 DNA 片段,请按以下步骤操作:对于位点 I的两条链中的每一条,提取与 3CMW 结构(化学计量学)中的C A (分别为 C B )结合的三个核苷酸的的RecA / DNA关联是3个核苷酸/单体); 请注意,确定目标核苷酸的残基数可能需要使用VMD对 3CMW 结构进行目视检查。钢绞线在现场II是从我们发表在阳模型拍摄等。(2015)( 。PDB文件是根据要求提供):选择一个RecA的单体和所结合的DNA。然后,使用PTOOLS Ç ommands (图1B),superpo本身在RecA的单体至Ç甲(分别Ç乙),计算相关联的变换矩阵用的叠加,并且该变换矩阵应用于所述DNA区段。最后,对于每个股数,生成一个 . PDB文件,命名为CA_x.pdb其中x = 1,2 ,或3,其包含C甲单体和其相关的ATP分子,镁离子,和DNA片段(一个或多个)。          2组装积木            ia使用PTools / Heligeom (参见图 2B)通过命令行构建具有 3CMW 几何特征的 6 单体长丝段:          $ ptools heligeom -n 6 -o BLOCK1.pdb -Z CA_x.pdb CB_x.pdb


IB在Python脚本到其中的PTOOLS包已经被导入,构建刚体对应于R的对象A,- [R乙,和BLOCK1(分别是RA,RB ,和BL1 )。选择BL1 的链 A并将其存储在Rigidbody对象B1 中。选择BL1 的链 F并将其存储在Rigidbody对象B6 中。叠加RA上以RB和输出对应的变换矩阵MAT1 (图2A) 。适用MAT1上至B6 ,从而产生单体B7邻接B6以下结合几何特性2REB(图2C)。叠加B1上至B7和计算变换矩阵MAT2 (图2D); 适用MAT2上于BL1 ,从而产生一个新的6 -单体灯丝段BL2相邻BL1 ,与最后部之间的结合的几何形状BL1和的第一单元BL2是2REB类型(图2E和2F)。         



图 2.复合长丝的构造。(A) 定义两种RecA-RecA绑定模式的构建块的示意图:(顶部)C A -C B类型和(底部)R A -R B类型;变换矩阵的计算MAT1 ,其涉及刚体对象RA和RB (箭头)。(B)施加的ħ eligeom命令行到积木Ç甲和C乙产生与C的接口6单元螺旋甲-C乙类型(步骤A2A) 。(C)产生的Rigidb ö DY B7从B6通过施加MAT1 ; B6和B7通过RA-RB接口交互。(D)将s B1转换为B7的mat2计算。(E)施加MAT2到所述BL1块。(F)最终结果(见 A.2.b 中的文本)。PTOOLS对象名称都写在蓝色使用时和红色定义的时候。该过程在Figure2_video.mp4的随附视频中进行了说明。


3细化构建块之间的连接                a通过用来自RA的等效区域替换Rigidbody B7的 N 端结构域(一个螺旋和相关接头,残基 1 至 37)来调整界面柔性基团。为了此结束,创建一个空的刚体对象BL2_adjusted 。选择残基编号为 38 到 156、165 到 193和213 到 333 的RA (非柔性区域)的 C α原子并将它们存储在Rigidbody SRA 中。对B7执行相同的选择,存储在SB7 中;叠加SRA上至SB7和输出的变换矩阵MAT3 。选择RA的 N 端结构域,残基 1 到 37,并将选择存储在NT 中;将B7 的第38 至 333 位残基储存在CB7 中;连锁店B,C,d,E ,和F的BL2在BL2_BtoF 。将mat3应用到NT并将结果存储在NT7 中。先后串联NT7 ,CB7 ,和BL2_BtoF到BL2_adjusted ; 将BL2_adjusted连接到BL1 。以 PDB 格式输出结构,命名为FINAL_ x.pdb ,其中 x = 1, 2, 3 是 DNA 链的数量。现在已经生成了三个最终结构。                  b以这样的方式处理最终结构,将 DNA 片段分组,用特定的链名标记它们,将 ATP 分子分组并更改其链名,并将蛋白质链从 A 命名为 L(图 2C,右);用ADP替换与链 F 结合的 ATP/Mg 2+ 。这可以蟒蛇/内完成PTOOLS脚本输出之前。上一段中的pdb文件或通过处理. pdb文件使用文本编辑器。                  4关闭每个文件FINAL_x.pdb 的限制最小化的 DNA 链,x = 1, 2, 3                IA使用配方1 ,大致重新定位的三核苷酸的DNA片段T3 ˚F从关联链3与单体F.此段呈现与单体ģ众多立体冲突以下在灯丝轴线取向的变化,连续到该FG界面几何形状的修改。方向的变化还导致T3 F和与单体 G 相关的链 3的 DNA 片段 T3 G之间的位点 II 连续性的重要断裂。              ib生成拓扑。使用« Automatic PSF builder » VMD 扩展的.psf文件。            ic从FINAL_x.pdb文件为 NAMD 最小化创建约束文件,方法是将« 1.00 »添加到约束原子的第 62 列(P 表示 DNA 链,CA 表示蛋白质);给定为脚本配方2下面可以用于此端。需要修改生成的文件FINAL_x_cstr.pdb以释放位于链s F 和 G之间的连接处的磷酸原子,以实现能量最小化,其中螺旋对称性已被破坏,为此将« 1.00 »替换为« 0.00 »特定的磷酸原子(每个 DNA 链释放三个磷酸原子)。              id使用NAMD2.10执行 5000 步共轭梯度能量最小化。这使得能够CLOS的URE所述DNA骨架通过根据拓扑文件恢复共价连接的原子之间的距离和释放如果任何小空间碰撞。            B长丝模型的稳定性和演化:分子动力学模拟                1模拟准备每个文件FINAL_x.pdb , x = 1, 2, 3                ia使用 VMD 扩展 «Add Solvation Box» 将结构浸入水箱中;使用 TIP3P 水模型和10的框填充值。              ib使用 VMD 扩展名“添加离子”将 Na +和 Cl -离子添加到水箱中;选择选项«中和并设置NaCl浓度为 0.15 mol /L»。            2使用NAMD2.10对三个系统中的每一个进行仿真                鉴于大尺寸的系统(约45万个原子)的,该步骤需要执行分布式计算机节点上,例如,在计算机中心。仿真遵循标准协议NAMD小号。


ia模拟条件:使用 CHARMM 27 力场,包括 CMAP 修正;使用周期性边界条件,使用粒子混乱 Ewald 方法来解释长程相互作用和范德华相互作用在 10 和 12 Å之间的平滑切换;使用 SHAKE 算法将时间步长设置为 2 fs;使用Langevin动力学方案和 Nose-Hoover Langevin活塞控制温度和压力。              IB模拟运行:执行5000个步骤的共轭梯度最小化,接着由渐进加热阶段高达300K,30纳秒长平衡相的,和一个100纳秒生产阶段。在加热和平衡过程中,对锉的 P 和 CA 原子设置约束,力常数从 0.5 减小到 0.05 kcal · mol -1 · Å -2 。在生产阶段不要使用约束。使用相同的条件对三链系统执行三个独立的模拟。            数据分析


以下的分析步骤的结果可以在找到小号upplemental我载文信息(SI)的博耶等人。(2015)。


1传统的 MD 分析。使用 VMD 工具检查分子动力学模拟轨迹期间能量和温度的稳定性。通过消除水分子来减小轨迹文件的大小。绘制根均的时间演变-平方波动(RM小号F)或偏差(RMSD)的系统中,在Cα原子结合的系统的值或选择的国家和地区:整个灯丝,灯丝中心,DNA中心(博耶等人., 2015, 图 SI-7) 。Ť o每个单体的波动从第分离OSE整个灯丝的,计算的RMSD和RMSF值沿着轨迹为每个单独的RecA单体,叠加局限于此单体后; 绘制所得 RMSD 平均值的演变,采用所有单体(Boyer等人,2015 年,图 SI-7);单独绘制对应于每个单体的 RMSF (Boyer等人,2015,图 SI-5)。使用VMD目视检查轨迹的最终结构,以选择感兴趣的原子或原子组,这些原子或原子组因界面修改而改变与其他组的接触;绘制这些距离的时间演变;计算在常规细丝相应的距离和在并入该值到积为水平线为基准(博耶等人,2015,图SI-3) 。                2丝状组件的特定 MD 分析。CA在各单体的水平lculate槽入口的宽度,如在博耶描述等。(2 015 )(材料小号和方法); 积为每个单体的宽度值的时间演变,并然后绘制在常规长丝测定值在博耶等。(2015 年),图 SI-2 。使用PTools实用程序,计算每个非终端接口的f NAT值沿轨迹的演变,其中f NAT是在给定快照中保存的起始结构中对接触的分数(接触被定义为来自每个相互作用单体的一对残基,它们的距离小于 5 Å,并且对接触仅限于界面的非柔性区域,如上面步骤 A3a 中所定义)(Boyer等人,2015,图 SI- 6)。                3 DNA分析。DNA 链之间相互作用的演变通过两种方式进行监测:通过测量每个快照的每对链之间最短的磷酸盐 - 磷酸盐链间距离(参见 Boyer等人,2015,图 SI-9 了解详细信息)或通过计算三个碱基对之间配对距离的时间演变,使用VMD通过目视检查选择(Boye r等人,20 15,图 4)。                笔记


1在 3CMW 中。PDB文件时,残基被编号为1至333的第一单体,1001至1333年,第二,2001至2333年的第三,等,这是可取的编辑文件CA.pdb和CB.pdb为了具有该范围内的残基编号的1-333。必须注意所有对. pdb文件尊重. pdb文件格式。        2在这项工作中生成的模型存放在ModelArchive https://www.modelarchive.org/ 中,代码为ma-900pk 、ma-l1kfl和ma-eaaa9 ,可在以下 URL 公开获取:      https://www. modelarchive.org/doi/10.5452/ [code ] (其中[code]是ma-900pk 、ma-l1kfl或ma-eaaa9 )。        食谱


1个的T3 ˚F在步骤A4A定义三核苷酸段可以被重新定位通过按照下面的命令,其中用于链3被“P链索引,”的T3 ˚F核苷酸编号16至在5 18 “ -3 ”方向,核苷酸编号15相关联用单体E ,和核苷酸19是T3的第一个核苷酸ģ中的5 '方向。核苷酸18和19之间产生的不连续骨干下面三个点限定了脚本,O,A,和乙,其中O位于核苷酸15原子P甲上原子O3'的核苷酸19,和B关于的原子P核苷酸18. T3 ˚F将由40旋转°围绕所述轴线(O型,U ),其中u是矢量OA和OB之间的归一化互产物:        $蟒蛇


>>>从ptools导入 *


>>>菲拉=刚体( “ FINAL_3.pdb”)


>>> strP = fila.select_chain_id ("P")。create_rigid ()


>>> t3f = strP.select_res_范围(15,18)。create_rigid ()


>>>原子=( strP.select_res_range (15,15) &    \


          strP.select_atom_类型(“P”))。create_rigid ()


>>> O = atom.get_coords(0)


>>>原子=( strP.select_res_range (19,19) &     \


          strP.select_atom_类型(“O3'”))。create_rigid ()


>>> A = atom.get_coords(0) >>>原子=( strP.select_res_range (18,18) &    \


          strP.select_atom_类型(“P”))。create_rigid ()


>>> B = atom.get_coords(0)


>>> OA = A - O


>>> OB = B - O


>>> u = Coord3D()


>>> ux = OA.y * OB.z - OA.z * OB.y


>>> uy = OA.z * OB.x - OA.x * OB.z


>>> uz = OA.x * OB.z - OA.z * OB.x


>>> u.normalize ()


>>> newt3f =刚体(T3F)


>>> newt3f.ab_rotate ( O,O+u,40*3.14/180.)


>>> write_ pdb ( newt3f,"new_T3F.pdb")


然后可以编辑文件FINAL_3.pdb以合并new_T3F.pdb代替T3 F。


2以下大号inux下命令可用于产生用于NAMD一个约束文件为了抑制P和Ca原子的能量最小化,在加热过程中的位移,和平衡(见步骤的开始A4C):        $ awk '$3 == "P" || $3 == "CA" { \


>          printf "%s 1.00%s\n" , substr ($0,1,61), substr ($ 0,67 )}; \


>          3美元!= "P" && $3 != "CA" ' FINAL_x.pdb > FINAL_x_cstr.pdb


此命令标记文件FINAL_x.pdb 的所有“ P ”和“ CA ”原子。为了最小化过程,以便能够关闭DNA骨架,从结区域磷酸盐原子必须通过替换“被释放1.00在列63”到66与“ 0.00 ”。例如,对于链 3,如果保留配方 1 中的符号,则将释放链“ P ”的15 至 20磷酸。


致谢


作者要感谢“倡议d ”卓越'的法国政府的计划提供资金[电机,ANR-11的LabX-0011-01]。原研纸从这个协议是衍生物通过博耶出版等。(2019)。我们感谢 Raquel de Miranda 协助准备描述RecA灯丝制备的视频。


利益争夺


作者声明他们已经没有竞争兴趣小号。


伦理


该协议提出这里没有使用任何人或动物受试者小号。


参考


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引用:Boyer, B., Laurent, B., Robert, C. H. and Prévost, C. (2021). Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom. Bio-protocol 11(14): e4097. DOI: 10.21769/BioProtoc.4097.
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