The use of fixed chunk regions and fragment jumps for domain insertion modeling.

<p>a) The structure of the domain insertion protein YahK (PDB code 1uuf) color-coded to indicate the regions that various client Movers act on. Gray indicates traditional torsion fragment insertion and loop closure, and green indicates torsion and β-strand fragment insertion. Blue and red are, respectively, the N- and C-terminal portions of the host domain. Those regions are fixed. b) A possible consensus fold tree (note that many fold trees are valid consensus fold trees depending upon the choices made at run time for cut placement, β-strand pairing choices, <i>etc</i>.). The two discontinuous chains of the host domain (color-coded as above) are fixed in their relative geometry by the jump that connects the C- and N-terminal regions directly. The insert is broken into multiple stretches (grey and green) by the jumps created for β-strand pairing (green).</p

The design of the broking mechanism.

<p>a) right: the central resources of a Rosetta protocol (blue) are acted upon by many independent Movers (green) in an uncontrolled fashion. Movers differ in the actions they perform on these resources, including configuration (green arrows) and sampling (black arrows). Left: A Broker layer (purple) receives requests from numerous clients (green) using a standard interface, and configures the core resources appropriately. Access is restricted to these resources using an access control framework, but requests invisibly “pass through” this layer to avoid interface differences. b) The Broker communicates with client Movers by receiving claims and responding with a passport. c) Client Movers (light green) convert user-specified configurations (brown) into convert developer-friendly claims (light purple, left) through the claiming interface. The Broker (dark purple) converts claims into specific, machine-readable needs, which are processed and returned to the client Mover as a DoF passport (light purple, right). d) The DoF access assignment behavior of the Broker when two clients request access to the same DoF. If one Mover claims exclusive and another claims must control or exclusive, broking fails, because it is not possible to satisfy both. If one claims exclusive and the other claims can control, only the Mover claiming exclusive receives access. If a Mover claims “does not control,” it never receives access. In all other cases, both Movers receive access. e) The procedure by which the conformation validates a modification to a DoF. The client Mover creates an unlock, which is shared by the conformation and the Mover. Then, whenever to the conformation change the DoF, the conformation checks latest active unlock to ensure the active Mover has access to the changing degrees of freedom.</p

A SnugDock-inspired antibody modeling protocol configuration.

<p>a) An antibody heavy (red) and light (blue) chain in complex with an antigen (green), which interacts with the antibody’s CDR loops (cyan). Call-outs identify the sampling procedures that are active on this structure using this protocol, and colors indicate the regions that each prodcedure targets: the gradient-based minimizer (cyan), loop closure (magenta), and fixed backbone docking (red, green, and blue) of antibody chains and antigen. Additionally, the explicitly-monitored centers of mass of each of the three polypeptide chains are indicated (blue, green, and red circles) and each is docked to a central reference point (grey circle). b) The fold tree that underlies the situation in (a). Each chain is docked via its center of mass virtual residue (red, blue, and green circles) to a central virtual residue (grey circle). The antigen and antibody regions outside of CDR loops are fixed, whereas the CDR loops, each of which is interrupted by a cut, are flexibly modeled by minimization and subjected to loop closure. The color of the line indicates where Mover active: red, green, and blue are docking Movers, magenta and cyan are loop closure and minimization, respectively, and grey is unmoved. c) The definition of the ResidueSelectors used in the body of the script XML script. Note that many residue selectors are created using Boolean logic operators depending on other ResidueSelectors, making alterations straightforward.</p

Multi-resolution constraints in a simple folding protocol.

<p>a) The crystal structure of ubiquitin is color- coded and annotated by the sampling procedures applied to each region. Green strands indicate regions of β-strand pair sampling, the blue region has fixed internal coordinates drawn from the native crystal structure, and grey and green are subject to fragment insertion using fragments from chemical shifts. b) One possible fold tree generated by the Broker to satisfy the constraints given in (a). Black pointing arrows represent jumps between β strands, which are represented by green block arrows. Breaks in the underlying black line indicate possible chain break locations. The fixed region is indicated by the blue rounded rectangle. c) The main part of a RosettaScripts XML script that implements this protocol. The full script is available in the supplement and the script along with all required files is available in the protocol capture.</p

RosettaMP directly extends the architecture of Rosetta3.

<p>Every Rosetta protocol requires at least these three main objects for modeling or design tasks (light blue): a Pose to a represent a biomolecule, a ScoreFunction to rank modeled structures and sequences, and Movers to sample new conformations of the Pose. RosettaMP directly extends this architecture (blue) by adding an element to the Pose representing the membrane bilayer, restructuring the original membrane ScoreFunction to rely on this membrane representation, and implementing a new set of Movers to sample the conformational search space available in the membrane bilayer.</p

An Integrated Framework Advancing Membrane Protein Modeling and Design

<div><p>Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design.</p></div

Assembly of symmetric protein complexes in the membrane using MPsymdock.

<p>(A) FoldTree representation of the homo-tetrameric KcsA potassium channel with the membrane residue (M) at the root (circled). The virtual residues V_1,i and V_2,i required for the symmetry machinery are described in the text. (B) Native structure in gray (PDB 1bl8) superimposed with the model from MPsymdock with the lowest interface score. The view is from the extracellular side of the membrane. (C) Membrane plane view of (B). (D) Interface score vs. backbone RMSD to the native structure for 1000 models of the KcsA potassium channel. The lowest scoring model, shown in (B) and (C), is indicated in red.</p

MPddG computes free energy changes upon mutation in the membrane environment (ΔΔG).

<p>(A) Outer membrane protein phospholipase A (OmpLA, PDB 1qd6) with its native alanine at position 210 in red at the center of the membrane. (B) Plot of RosettaMP-calculated fixed-backbone ΔΔ<i>G</i>s versus experimentally measured values of Moon & Fleming for variants at position 210 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004398#pcbi.1004398.ref050" target="_blank">50</a>]. Proline is off-scale (ΔΔ<i>G</i>_<i>pred</i> = 193.2 REU) due to incompatible backbone torsions yielding ring closure penalties. (C) Outer membrane protein A (OmpA, PDB 1qjp) with aromatic residues mutated to alanine at various interfacial positions. (D) Plot of RosettaMP-calculated ΔΔ<i>G</i>s versus experimentally measured values of Hong & Tamm [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004398#pcbi.1004398.ref051" target="_blank">51</a>]. The mutation W15A is off-scale (ΔΔ<i>G</i>_pred = -43.0 REU) due to the loss of repulsive interactions upon mutation to alanine. Both (B) and (D) include a line for <i>y</i> = <i>x</i>.</p

MPrelax for high-resolution refinement of a membrane protein.

<p>(A) FoldTree representation for the MPrelax protocol with the residue closest to the center-of-mass of the protein being at the root of the FoldTree (circled X). The membrane residue (M) is attached via a flexible jump edge (dashed arrow). Protein chains are shown as gray boxes with N- and C- termini marked and peptide edges shown as solid arrows. (B) Rosetta total score vs. backbone RMSD to the crystal structure for 1000 models of meta-rhodopsin. Models in blue are created with the original membrane relax protocol of RosettaMembrane; models in red are created with MPrelax. (C) Crystal structure of meta-rhodopsin in gray (PDB 3pxo) superimposed with the lowest scoring models from both the original RosettaMembrane protocol (blue) and the MPrelax protocol (red).</p

Rosetta membrane energy terms used by RosettaMP.

<p>¹ Scope of individual energy terms. 1b indicates a per-residue or per-atom score (one-body), 2b indicates a two-body score, z indicates the score is dependent upon depth in the membrane bilayer, cd indicates the score depends on context (typically the number of surrounding residues), and ws is a score based on the whole structure.</p><p>Rosetta membrane energy terms used by RosettaMP.</p