Excited-State Dynamics of a CRABPII-Based Microbial Rhodopsin Mimic.

Microbial rhodopsin, a pivotal photoreceptor protein, has garnered widespread application in diverse fields such as optogenetics, biotechnology, biodevices, etc. However, current microbial rhodopsins are all transmembrane proteins, which both complicates the investigation on the photoreaction mechanism and limits their further applications. Therefore, a specific mimic for microbial rhodopsin can not only provide a better model for understanding the mechanism but also can extend the applications. The human protein CRABPII turns out to be a good template for design mimics on rhodopsin due to the convenience in synthesis and the stability after mutations. Recently, Geiger et al. designed a new CRABPII-based mimic M1-L121E on microbial rhodopsin with the 13-cis, syn (13C) isomerization after irradiation. However, it still remains a question as to how similar it is compared with the natural microbial rhodopsin, in particular, in the aspect of the photoreaction dynamics. In this article, we investigate the excited-state dynamics of this mimic by measuring its transient absorption spectra. Our results reveal that there are two components in the solution of mimic M1-L121E at pH 8, known as protonated Schiff base (PSB) and unprotonated Schiff base (USB) states. In both states, the photoreaction process from 13-cis, syn(13C) to all-trans,anti (AT) is faster than that from the inverse direction. In addition, the photoreaction process in the PSB state is faster than that in the USB state. We compared the isomerization time of the PSB state to that of microbial rhodopsin. Our findings indicate that M1-L121E exhibits behaviors similar to those of microbial rhodopsins in the general pattern of PSB isomerization, where the isomerization from 13C to AT is much faster than its inverse direction. However, our results also reveal significant differences in the excited-state dynamics of the mimic relative to the native microbial rhodopsin, including the slower PSB isomerization rates as well as the unusual USB photoreaction dynamics at pH = 8. By elucidating the distinctive characteristics of mimics M1-L121E, this study enhances our understanding of microbial rhodopsin mimics and their potential applications.

Degradation protection and enhanced biocompatibility of Mg alloys pretreated with plasma proteins.

After implantation of the Mg alloy in the human body, the adsorption of plasma protein on surface will cause a series of cell reactions and affect the degradation of Mg alloys. Herein, in vitro biological reactions of the ZK60 and AZ31 Mg alloys are analyzed in plasma protein environment. Combined with mass spectrometry analysis of the type of adsorbed proteins, it is shown that proteins such as fibrinogen, vitronectin, fibronectin, and prothrombin are prone to get adsorbed on the surface of the alloys than other proteins, leading to the promotion of MG63 cell adhesion and proliferation. The effect of selected proteins (fibrinogen, fibronectin, and prothrombin) on degradation of ZK60 and AZ31 Mg alloys is investigated using immersion tests. The degradation of AZ31 Mg alloy is significantly restrained with the presence of proteins. This is due to the protein adsorption effect on the sample surface. The molecular dynamics simulation results indicate that both fibrinogen and fibronectin tend to adsorb onto the AZ31 rather than ZK60, forming a stable protein layer on the AZ31 Mg alloy retarding the degradation of the samples. As to ZK60 alloy, the addition of protein inhibits the degradation in the short term, however, the degradation increases after a long time of immersion. This phenomenon is particularly pronounced in fibronectin solution.

Crystal structures of MHC class I complexes reveal the elusive intermediate conformations explored during peptide editing.

Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between numerous conformational states and these motions support peptide sampling. To date, MHC I intermediates are largely uncharacterized experimentally and remain elusive. Here, we present x-ray crystal structures of HLA-B8 loaded with 20mer peptides that show pronounced distortions at the N-terminus of the groove. Long stretches of N-terminal amino acid residues are missing in the electron density maps creating an open-ended groove. Our structures also reveal highly unusual features in MHC I-peptide interaction at the N-terminus of the groove. Molecular dynamics simulations indicate that the complexes have varying degrees of conformational flexibility in a manner consistent with the structures. We suggest that our structures have captured the remarkable molecular dynamics of MHC I-peptide interaction. The visualization of peptide-dependent conformational motions in MHC I is a major step forward in our conceptual understanding of dynamics in high-affinity peptide selection.

From Feynman's ratchet to time crystalline molecular motors.

Cats have an instinctive ability to use the connection governing parallel transport in the space of shapes to land safely on their feet. Here, we argue that the concept of connection, which is extensively used in general relativity and other parts of theoretical physics, also explains the impressive performance of molecular motors by enabling molecules to evade the conclusions of Feynman's ratchet-and-pawl analysis. First, we demonstrate the emergence of directed rotational motion from shape changes, which is independent of angular momentum. Then, we computationally design knotted polyalanine molecules and demonstrate the organization of individual atom thermal vibrations into collective rotational motion, which is independent of angular momentum. The motion occurs effortlessly even in ambient water and can be further enhanced through spontaneous symmetry breaking, rendering the molecule an effective theory time crystal. Our findings can be experimentally verified via nuclear magnetic resonance measurements and hold practical potential for molecular motor design and engineering.

De Novo Design of a ß-Helix Tau Protein Scaffold: An Oligomer-Selective Vaccine Immunogen Candidate for Alzheimer's Disease.

Tau pathology is associated with many neurodegenerative disorders, including Alzheimer's disease (AD), where the spatio-temporal pattern of tau neurofibrillary tangles strongly correlates with disease progression, which motivates therapeutics selective for misfolded tau. Here, we introduce a new avidity-enhanced, multi-epitope approach for protein-misfolding immunogen design, which is predicted to mimic the conformational state of an exposed epitope in toxic tau oligomers. A predicted oligomer-selective tau epitope 343KLDFK347 was scaffolded by designing a ß-helix structure that incorporated multiple instances of the 16-residue tau fragment 339VKSEKLDFKDRVQSKI354. Large-scale conformational ensemble analyses involving Jensen-Shannon Divergence and the embedding depth D showed that the multi-epitope scaffolding approach, employed in designing the ß-helix scaffold, was predicted to better discriminate toxic tau oligomers than other "monovalent" strategies utilizing a single instance of an epitope for vaccine immunogen design. Using Rosetta, 10,000 sequences were designed and screened for the linker portions of the ß-helix scaffold, along with a C-terminal stabilizing α-helix that interacts with the linkers, to optimize the folded structure and stability of the scaffold. Structures were ranked by energy, and the lowest 1% (82 unique sequences) were verified using AlphaFold. Several selection criteria involving AlphaFold are implemented to obtain a lead-designed sequence. The structure was further predicted to have free energetic stability by using Hamiltonian replica exchange molecular dynamics (MD) simulations. The synthesized ß-helix scaffold showed direct binding in surface plasmon resonance (SPR) experiments to several antibodies that were raised to the structured epitope using a designed cyclic peptide. Moreover, the strength of binding of these antibodies to in vitro tau oligomers correlated with the strength of binding to the ß-helix construct, suggesting that the construct presents an oligomer-like conformation and may thus constitute an effective oligomer-selective immunogen.

Long-Time Dynamics of Selected Molecular-Motor Components Using a Physics-Based Coarse-Grained Approach.

Molecular motors are essential for the movement and transportation of macromolecules in living organisms. Among them, rotatory motors are particularly efficient. In this study, we investigated the long-term dynamics of the designed left-handed alpha/alpha toroid (PDB: 4YY2), the RBM2 flagellum protein ring from Salmonella (PDB: 6SD5), and the V-type Na+-ATPase rotor in Enterococcus hirae (PDB: 2BL2) using microcanonical and canonical molecular dynamics simulations with the coarse-grained UNRES force field, including a lipid-membrane model, on a millisecond laboratory time scale. Our results demonstrate that rotational motion can occur with zero total angular momentum in the microcanonical regime and that thermal motions can be converted into net rotation in the canonical regime, as previously observed in simulations of smaller cyclic molecules. For 6SD5 and 2BL2, net rotation (with a ratcheting pattern) occurring only about the pivot of the respective system was observed in canonical simulations. The extent and direction of the rotation depended on the initial conditions. This result suggests that rotatory molecular motors can convert thermal oscillations into net rotational motion. The energy from ATP hydrolysis is required probably to set the direction and extent of rotation. Our findings highlight the importance of molecular-motor structures in facilitating movement and transportation within living organisms.

Unusual crystal structures of MHC class I complexes reveal the elusive intermediate conformations explored during peptide editing in antigen presentation.

Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between conformational states as they sample peptides for potential ligands. To date, MHC I intermediates are largely uncharacterized experimentally and remain elusive. We present x-ray crystal structures of HLA-B8 loaded with 20mer peptides that show significant conformational heterogeneity at the N-terminus of the groove. Long stretches of N-terminal residues were missing in the electron density maps creating an unstructured and widely open-ended groove. Our structures also revealed highly unusual features in MHC I and peptide conformations, and in MHC I-peptide interaction at the N-terminus of the groove. Molecular dynamics simulations showed that the complexes have varying degrees of flexibility in a manner consistent with the structures. We suggest that our structures represent transient substates explored by MHC I molecules during peptide editing. The visualization of peptide-dependent conformational flexibility in MHC I groove is a major step forward in our conceptual understanding of peptide repertoire development in antigen presentation. Our study also raises questions about the role of the N-terminus of the groove in peptide editing.

In silico method for identifying the key residues in a knotted protein: with MJ0366 as an example.

The knotted proteins are a class of rare but biologically important proteins, due to the special topology of their native structure. Here we present a simple in silico method to identify the key residues for knotting and unknotting in a knotted protein, using the trefoil protein MJ0366 as an example. We first simulate the folding process via the annealing molecular dynamics (AMD) simulations in the coarse-grained "Go"-like model. From the folding trajectories, we monitor the knotting process using the quantity "length of knot tails". In the meantime, we analyze the evolution of the local geometry of the Cα trace with the help of the Discrete Frenet Frame (DFF). We identify the key residues by correlating the local geometry at each residue with the variable "length of knot tails" in the folding process, where a higher correlation coefficient indicates that the residue is more important for knotting. We validate our method by comparing with the experimental results in the literature. With the same method, we further predict the key residues for unknotting MJ0366 using the AMD simulations in both the coarse-grained "Go"-like model and all-atom (AA) force field model, respectively. We find that the key residues for unknotting are partially overlapped with those for knotting, indicating that the pathways for unknotting and knotting are generally similar except for the existence of some non-native contact interactions in the unknotting process. This in silico method can provide a new insight for understanding the knotting and unknotting processes of a knotted protein.

Excited-state dynamics of all-trans protonated retinal Schiff base in CRABPII-based rhodopsin mimics.

The rhodopsin mimic is a chemically synthetized complex with retinyl Schiff base (RSB) formed between protein and the retinal chromophore that can mimic the natural rhodopsin-like protein. The artificial rhodopsin mimic is more stable and designable than the natural protein and hence has wider uses in photon detection devices. The mimic structure RSB, like the case in the actual rhodopsin-like protein, undergoes isomerization and protonation throughout the photoreaction process. As a result, understanding the dynamics of the RSB in the photoreaction process is critical. In this study, the ultrafast transient absorption spectra of three mutants of the cellular retinoic acid-binding protein II-based rhodopsin mimic at acidic environment were recorded, from which the related excited-state dynamics of the all-trans protonated RSB (AT-PRSB) were investigated. The transient fluorescence spectra measurements are used to validate some of the dynamic features. We find that the excited-state dynamics of AT-PRSB in three mutants share a similar pattern that differs significantly from the dynamics of 15-cis PRSB of the rhodopsin mimic in neutral solution. By comparing the dynamics across the three mutants, we discovered that the aromatic residues near the ß-ionone ring structure of the retinal may help stabilize the AT-PRSB and hence slow down its isomerization rate. The experimental results provide implications on designing a rhodopsin-like protein with significant infrared fluorescence, which can be particularly useful in the applications in biosensing or bioimaging in deeper tissues.

Ultralow-threshold green fluorescent protein laser based on high Q microbubble resonators.

Biological lasers have attracted vast attention because of their potential medical application prospects, especially the low threshold biological laser that can be used for ultrasensitive biological detection while leaving the luminous gain medium undamaged by the pump light. By coupling the low concentration green fluorescent protein (GFP) solution with a high Q whispering gallery mode microbubble resonator, we managed to fabricate a miniature GFP laser with the lowest threshold and highest Q value compared to any known type of the GFP laser. The threshold energy is as low as 380 fJ, two orders of magnitude lower than any type of GFP laser at present. The Q value of the optical cavity in this biological laser is 5.3 × 107, two orders higher than the highest Q value of GFP lasers. We further confirmed the long-term stability of the working characteristics of GFP laser. It can work well nearly a month in temperature 3-4°C. Finally, we measured the effects of different concentrations of fluorescent protein on laser threshold. The data show that this biological laser can be used for highly sensitive detection of GFP concentration, which is particularly useful when the GFP is used as tracers.

Rational Generation of Monoclonal Antibodies Selective for Pathogenic Forms of Alpha-Synuclein.

Misfolded toxic forms of alpha-synuclein (α-Syn) have been implicated in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The α-Syn oligomers and soluble fibrils have been shown to mediate neurotoxicity and cell-to-cell propagation of pathology. To generate antibodies capable of selectively targeting pathogenic forms of α-Syn, computational modeling was used to predict conformational epitopes likely to become exposed on oligomers and small soluble fibrils, but not on monomers or fully formed insoluble fibrils. Cyclic peptide scaffolds reproducing these conformational epitopes exhibited neurotoxicity and seeding activity, indicating their biological relevance. Immunization with the conformational epitopes gave rise to monoclonal antibodies (mAbs) with the desired binding profile showing selectivity for toxic α-Syn oligomers and soluble fibrils, with little or no reactivity with monomers, physiologic tetramers, or Lewy bodies. Recognition of naturally occurring soluble α-Syn aggregates in brain extracts from DLB and MSA patients was confirmed by surface plasmon resonance (SPR). In addition, the mAbs inhibited the seeding activity of sonicated pre-formed fibrils (PFFs) in a thioflavin-T fluorescence-based aggregation assay. In neuronal cultures, the mAbs protected primary rat neurons from toxic α-Syn oligomers, reduced the uptake of PFFs, and inhibited the induction of pathogenic phosphorylated aggregates of endogenous α-Syn. Protective antibodies selective for pathogenic species of α-Syn, as opposed to pan α-Syn reactivity, are expected to provide enhanced safety and therapeutic potency by preserving normal α-Syn function and minimizing the diversion of active antibody from the target by the more abundant non-toxic forms of α-Syn in the circulation and central nervous system.

In silico study on the effects of disulfide bonds in ORF8 of SARS-CoV-2.

The COVID-19 epidemic, caused by virus SARS-CoV-2, has turned into a pandemic and threatened everyone's health for the past two years. In SARS-CoV-2, ORF8 is one of the most important accessory proteins with a role in immune modulation. There are multiple disulfide bonds in the wild type (WT) ORF8. Here, we present an in silico study on the effects of the disulfide bonds in ORF8 on the aspects of the structural properties and binding properties with the human leukocyte antigen (HLA-A). We first define five possible states for ORF8 with different disulfide bond reduction schemes. For each state, we collect the conformational ensemble using molecular dynamics (MD) simulations in an explicit solvent. From the analysis of the structural properties, we find that the reduction of the disulfide bonds has small effects on the global properties but much larger effects on the ORF8-specific region that is located on the surface of the ORF8 dimer. Interestingly, we find that the dimer does not break into two monomers at room temperature even if all the disulfide bonds get reduced. Further, we investigate the role of the disulfide bonds in the interactions with the human leukocyte antigen (HLA) by performing docking between HLA-A and the conformational ensembles of ORF8 in different states. We give predictions on the preferred binding sites for each state and validate the predictions for the WT dimer with the experimental data on epitopes. In the end, we evaluate the stability of the complexes formed between HLA-A and ORF8 in each state using MD simulations. Our observations can provide inspiration for inhibitor/drug design against ORF8 based on the binding pathway with HLA-A.

Optimizing Epitope Conformational Ensembles Using α-Synuclein Cyclic Peptide "Glycindel" Scaffolds: A Customized Immunogen Method for Generating Oligomer-Selective Antibodies for Parkinson's Disease.

Effectively presenting epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. To this end, we computationally modeled scaffolded epitopes in cyclic peptides by inserting/deleting a variable number of flanking glycines ("glycindels") to best mimic a misfolding-specific conformation of an epitope of α-synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We screen and rank the cyclic peptide scaffolds of α-synuclein in silico based on their ensemble overlap properties with the fibril, oligomer-model and isolated monomer ensembles. We present experimental data of seeded aggregation that support nucleation rates consistent with computationally predicted cyclic peptide conformational similarity. We also introduce a method for screening against structured off-pathway targets in the human proteome by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. Different cyclic peptide scaffolds with variable numbers of glycines are predicted computationally to have markedly different conformational ensembles. Ensemble comparison and overlap were quantified by the Jensen-Shannon divergence and a new measure introduced here, the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble and which may be a more useful measure in developing immunogens that confer conformational selectivity to an antibody.

First Principles Calculation of Protein-Protein Dimer Affinities of ALS-Associated SOD1 Mutants.

Cu,Zn superoxide dismutase (SOD1) is a 32 kDa homodimer that converts toxic oxygen radicals in neurons to less harmful species. The dimerization of SOD1 is essential to the stability of the protein. Monomerization increases the likelihood of SOD1 misfolding into conformations associated with aggregation, cellular toxicity, and neuronal death in familial amyotrophic lateral sclerosis (fALS). The ubiquity of disease-associated mutations throughout the primary sequence of SOD1 suggests an important role of physicochemical processes, including monomerization of SOD1, in the pathology of the disease. Herein, we use a first-principles statistical mechanics method to systematically calculate the free energy of dimer binding for SOD1 using molecular dynamics, which involves sequentially computing conformational, orientational, and separation distance contributions to the binding free energy. We consider the effects of two ALS-associated mutations in SOD1 protein on dimer stability, A4V and D101N, as well as the role of metal binding and disulfide bond formation. We find that the penalty for dimer formation arising from the conformational entropy of disordered loops in SOD1 is significantly larger than that for other protein-protein interactions previously considered. In the case of the disulfide-reduced protein, this leads to a bound complex whose formation is energetically disfavored. Somewhat surprisingly, the loop free energy penalty upon dimerization is still significant for the holoprotein, despite the increased structural order induced by the bound metal cations. This resulted in a surprisingly modest increase in dimer binding free energy of only about 1.5 kcal/mol upon metalation of the protein, suggesting that the most significant stabilizing effects of metalation are on folding stability rather than dimer binding stability. The mutant A4V has an unstable dimer due to weakened monomer-monomer interactions, which are manifested in the calculation by a separation free energy surface with a lower barrier. The mutant D101N has a stable dimer partially due to an unusually rigid ß-barrel in the free monomer. D101N also exhibits anticooperativity in loop folding upon dimerization. These computational calculations are, to our knowledge, the most quantitatively accurate calculations of dimer binding stability in SOD1 to date.

Local topology and bifurcation hot-spots in proteins with SARS-CoV-2 spike protein as an example.

Novel topological methods are introduced to protein research. The aim is to identify hot-spot sites where a bifurcation can alter the local topology of the protein backbone. Since the shape of a protein is intimately related to its biological function, a substitution that causes a bifurcation should have an enhanced capacity to change the protein's function. The methodology applies to any protein but it is developed with the SARS-CoV-2 spike protein as a timely example. First, topological criteria are introduced to identify and classify potential bifurcation hot-spot sites along the protein backbone. Then, the expected outcome of asubstitution, if it occurs, is estimated for a general class of hot-spots, using a comparative analysis of the surrounding backbone segments. The analysis combines the statistics of structurally commensurate amino acid fragments in the Protein Data Bank with general stereochemical considerations. It is observed that the notorious D614G substitution of the spike protein is a good example of a bifurcation hot-spot. A number of topologically similar examples are then analyzed in detail, some of them are even better candidates for a bifurcation hot-spot than D614G. The local topology of the more recently observed N501Y substitution is also inspected, and it is found that this site is proximal to a different kind of local topology changing bifurcation.

Field-effect at electrical contacts to two-dimensional materials.

The inferior electrical contact to two-dimensional (2D) materials is a critical challenge for their application in post-silicon very large-scale integrated circuits. Electrical contacts were generally related to their resistive effect, quantified as contact resistance. With a systematic investigation, this work demonstrates a capacitive metal-insulator-semiconductor (MIS) field-effect at the electrical contacts to 2D materials: The field-effect depletes or accumulates charge carriers, redistributes the voltage potential, and gives rise to abnormal current saturation and nonlinearity. On one hand, the current saturation hinders the devices' driving ability, which can be eliminated with carefully engineered contact configurations. On the other hand, by introducing the nonlinearity to monolithic analog artificial neural network circuits, the circuits' perception ability can be significantly enhanced, as evidenced using a coronavirus disease 2019 (COVID-19) critical illness prediction model. This work provides a comprehension of the field-effect at the electrical contacts to 2D materials, which is fundamental to the design, simulation, and fabrication of electronics based on 2D materials. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material (results of the simulation and SEM) is available in the online version of this article at 10.1007/s12274-021-3670-y.

Comparison of the force fields on monomeric and fibrillar PHF6 of tau protein.

The hexapeptide 306VQIVYK311 (PHF6) plays an important role in the aggregation of Tau protein, which is a hallmark of the Alzheimer's disease (AD). In this article, we systematically compare the effects of eight popular all-atom force fields on the monomeric and fibrillar PHF6 in the molecular dynamics (MD) simulations, which could be helpful in the computer-aided drug design against PHF6. We show that the fibrillar PHF6 prefers ß-strand-like structures in all the force fields while the monomer has different structural preferences depending on the force fields. The interactions for stabilizing the fibril are further investigated. In the end, according to the interactions revealed by NMR and the stability of the fibril in the literature, we benchmark the force fields.

A method for systematically ranking therapeutic drug candidates using multiple uncertain screening criteria.

Multiple different screening tests for candidate leads in drug development may often yield conflicting or ambiguous results, sometimes making the selection of leads a nontrivial maximum-likelihood ranking problem. Here, we employ methods from the field of multiple criteria decision making (MCDM) to the problem of screening candidate antibody therapeutics. We employ the SMAA-TOPSIS method to rank a large cohort of antibodies using up to eight weighted screening criteria, in order to find lead candidate therapeutics for Alzheimer's disease, and determine their robustness to both uncertainty in screening measurements, as well as uncertainty in the user-defined weights of importance attributed to each screening criterion. To choose lead candidates and measure the confidence in their ranking, we propose two new quantities, the Retention Probability and the Topness, as robust measures for ranking. This method may enable more systematic screening of candidate therapeutics when it becomes difficult intuitively to process multi-variate screening data that distinguishes candidates, so that additional candidates may be exposed as potential leads, increasing the likelihood of success in downstream clinical trials. The method properly identifies true positives and true negatives from synthetic data, its predictions correlate well with known clinically approved antibodies vs. those still in trials, and it allows for ranking analyses using antibody developability profiles in the literature. We provide a webserver where users can apply the method to their own data: http://bjork.phas.ubc.ca.

Autonomous topological time crystals and knotty molecular motors.

We show that topology is a very effective tool, to construct classical Hamiltonian time crystals. For this we numerically analyze a general class of time crystalline Hamiltonians that are designed to model the dynamics of molecular closed strings. We demonstrate how the time crystalline qualities of a closed string are greatly enhanced when the string becomes knotted. The Hamiltonians that we investigate include a generalized Kratky-Porod wormlike chain model in combination with long range Coulomb and Lennard-Jones interactions. Such energy functions are commonplace in coarse grained molecular modeling. Thus we expect that physical realizations of Hamiltonian time crystals can be constructed in terms of knotted ring molecules.

Recovering the Missing Regions in Crystal Structures from the Nuclear Magnetic Resonance Measurement Data Using Matrix Completion Method.

Based on matrix completion algorithm, we proposed a simple method to recover the missing regions in the X-ray crystal structures using the corresponding nuclear magnetic resonance (NMR) measurement data for the proteins with both X-ray and NMR experimental data deposited in Protein Data Bank (PDB). By selecting 10 test proteins deposited in PDB and comparing with the standard MODELLER results from the root-mean-square deviation and MolProbity aspects, we validated that our method can provide a better protein structure model, which combines both X-ray crystallographic structure data and NMR data together than MODELLER algorithm. This method is particularly useful for building the initial structures in Molecular Dynamics when studying the protein folding process.