Robetta Server Interview Questions & Answers

Question 1. What Is Robetta?

Answer :

Robetta is a full-chain protein structure prediction server. It parses protein chains into putative domains with the Ginzu protocol, and models those domains either by homology modeling or by ab initio modeling. Other services of the server, in addition to Domain Parsing and 3-D Modeling, include Fragment Library generation and Interface Alanine Scanning.

Question 2. What Are Fragment Libraries?

Answer :

Fragment Libraries are the pieces of experimentally determined structures that Rosetta uses to guide the search of conformational space when predicting structures using the ab initio protocol, as well as longer loop conformations in homology models.

Question 3. What Is Interface Alanine Scanning?

Answer :

Interface Alanine Scanning attempts to estimate the energetic contribution to the binding free energy provided by each residue at a protein-protein interface. Briefly, interface alanine scanning uses a simple physical model to score a series of protein-protein interfaces in which contact residues are individually replaced with alanine. After each computational alanine mutation, the resulting binding energy is calculated.

The input consists of a three-dimensional structure of a protein-protein complex; output is a list of "hot spots," or amino acid side chains that are predicted to significantly destabilize the interface when mutated to alanine, analogous to the results of experimental alanine-scanning mutagenesis. 79% of hot spots and 68% of neutral residues were correctly predicted in a test of 233 mutations in 19 protein-protein complexes. A single interface can be analyzed in minutes. The computational methodology has been validated by the successful design of protein interfaces with new specificity and activity and has yielded new insights into the mechanisms of receptor specificity and promiscuity in biological systems.

Question 4. What Is Ginzu?

Answer :

Ginzu is a protocol that attempts to determine the regions of a protein chain that will fold into globular units, called "domains". It scans the protein chain sequence with successively less confident methods of detection to determine any homologs with experimentally determined structures, starting with PDB-BLAST, and followed by the more remote fold-detection method HHSEARCH. After any homologs are identified, a search of remaining regions is done with HMMER against the Pfam-A protein family database.

Lastly, the PSI-BLAST multiple sequence alignment is used to assign regions of increased likelihood of possessing a contiguous domain based on sequence clusters. The final step consists of selecting cut-points between the domains (and possibly defining new domains based on the strongest cutpoints for any remaining long stretches of the sequence that have not already matched a homolog with a structure or Pfam-A) using the PSI-BLAST MSA.

Question 5. How Do I Submit A Sequence To The Structure Prediction Server?

Answer :

To submit a sequence to the structure server, you must be a registered user. To register, click here. If you are a registered user, go to the Structure Server submission form and do the following:

• Select a prediction type.
• Enter your username or registered email address.
• Enter a target name for your sequence.
• Paste your fasta sequence in the text area or upload your fasta file.
• Fill out optional fields if desired.
• Click the Submit button.

Question 6. How Do I Submit A Sequence To The Fragment Server?

Answer :

To submit a sequence to the fragment server, you must be a registered user. To register, click here. If you are a registered user, go to the Fragment Server submission form and do the following:

• Enter your username or registered email address.
• Enter a target name for your sequence.
• Paste your fasta sequence in the text area or upload your fasta file.
• Complete optional fields if desired. If you are uploading constraints data, be sure the formats are correct to prevent errors and wasted processor time.
• Click the Submit button.

Question 7. How Do I Submit A Sequence To The Interface Alanine Scanning Server?

Answer :

To submit a sequence to the interface alanine scanning server, you must be a registered user. To register, click here. If you are a registered user, go to the Alanine Scanning Server submission form and do the following:

• Enter your username or registered email address.
• Enter a job name for your sequence.
• Upload your protein complex file (must be PDB format).
• Define the interface for alanine scanning by entering the chain ID's (as in the complex; case sensitive) involved in the interface and the interface partners to which they belong.
• Optional: If you want results for specific interface side-chains, you can upload a Mutations List. Click here for file format. If there is a format error, all interface side-chains will be considered.
• Click the Submit button.

Question 8. How Do I Remove My Job From The Structure Prediction Server?

Answer :

To remove one or more of your jobs from the structure prediction server, follow these instructions:

• Login with your username and password.
• Go to the structure prediction queue table.
• Select one or more checkboxes (far left column under "x") for jobs that you own and would like to remove.
• Click the "Update Job(s)" button on the bottom of the page.
• All data will be lost, so be sure you retrieve your data before doing this.
• Jobs may be removed one week after they complete to conserve disk space.

Question 9. How Long Does A Job Take To Process?

Answer :

Both Rosetta ab initio and comparative modeling must generate ensembles of decoys before selecting models from amongst the ensemble. This process takes a considerable amount of computational resources, and therefore one can expect that, once your job has finished the Ginzu step and entered the ensemble generation, each domain will take a few hours (e.g. a 150 residue domain will take about 4 hours). Other long waits of up to 12 hours may occur during the initial Ginzu step.

Question 10. Why Am I Only Allowed One Job At A Time?

Answer :

Regrettably, due to the length of time it takes (on average) for jobs to complete, public users of the Robetta server are limited to one job at a time. We hope that this will prevent exceptionally long queues from occuring.

Question 11. How Much Time Do I Have Before My Job Is Removed And My Data Is Lost?

Answer :

We may remove domain and stucture prediction jobs and all their data one week after the date of completion to free disk space. This will be done at our own descretion.

Question 12. Can I Run Rosetta Myself?

Answer :

Due to hardware resource limitations, we suggest that if you wish to run a large number of predictions and have ample hardware of your own, that you obtain the Rosetta suite of programs themselves (which are freely available to academic users by license, and may be commercially licensed as well).

Question 13. Why Are All My Results Visible To Everyone?

Answer :

As a public resource, we feel that the free-flow of information is important to maintain. Additionally, we cannot take responsibility for providing the security necessary to protect users' results. Lastly, there may be more than one person who is interested in a particular target, and we want to avoid duplicate jobs.

Question 14. How Do I Configure My Browser To View Structures With Rasmol?

Answer :

Rasmol is a popular molecular graphics software package. If you do not have Rasmol, click here.

In theory, you should be able to configure a web browser to use Rasmol as a helper application to view PDB structures through the internet by setting the appropriate MIME type. However, it is difficult, if not impossible, to set up Internet Explorer to do this. Therefore, we recommend using a Netscape browser and the following instructions.

To configure your browser to view PDB structures, do the following:

• Go to your browser's Applications Preferences.
• Add a new file type configured as:
• Description: PDB files
• MIME Type: chemical/x-pdb
• Suffixes: .pdb,.ent
• Application: xterm -e rasmol -pdb %s

To configure your browser to view Rasmol scripts, do the following:

• Go to your browser's Applications Preferences.
• Add a new file type configured as:
• Description: RasMol scripts
• MIME Type: application/x-rasmol
• Suffixes: (leave blank)
• Application: xterm -e rasmol -script %s

In the examples above, we use 'xterm -e rasmol' as the helper application command that launches Rasmol in an xterm terminal window (Unix/Linux platforms). You may substitute this with 'rasmac' or 'raswin.exe' depending on what platform you are using.

Question 15. Why Do I Get More Than One Model?

Answer :

There are several models that the Robetta server produces when you submit a sequence. If Robetta determines that there is more than one domain (or if an ab initio portion of the sequence is too big to be modeled by the ab initio protocol), Robetta breaks up the query into putative domains and models each of them separately. After doing so, it assembles the models into one contiguous chain. This means you can examine your models either as a complete chain, or by clicking on the domain number in the Ginzu domain info box, you can examine the results for each individual domain.

Within each domain, there are several models. In the case of ab initio predictions, the models are the cluster centers of the most populated clusters, with the exception of the last model, which is the lowest energy decoy that was not a member of the previously represented clusters. In the case of homology modeling predictions, the second model is the model produced by the default K*Sync alignment, with the first, third, fourth, and fifth selected from the decoy ensemble by various energy discrimination methods. In the case of twilight-zone reliability parent detections, the first 5 models are homology modeled, and models six-ten modeled using the de novo protocol.

Question 16. Are There Any Issues With Long Sequences?

Answer :

The de novo protocol suffers, as do all such methods, from a limitation in the ability to sample conformations available to the protein. Larger targets that are high contact-order are more difficult to sample in a reasonable amount of computer time, and probably require much larger decoy ensembles than the Robetta server can afford to generate. Therefore, we impose a de novo domain size limit of about 200 residues, which is clearly often incorrect, but necessary. It is hoped that in such cases, features of the target are still captured by the models.

There is additionally a limit on the length of the full chain of about 650 residues, so that the independently modeled domains may be assembled into a contiguous chain.

Question 17. Are There Any Issues With Short Sequences?

Answer :

The Fragment Library used by the Rosetta folding program is generated by using residue substitution profiles with PSI-BLAST to find similar fragment profiles. Very short sequences (less than about 40 residues) are difficult to detect sequence homologs for with reliable confidence with PSI-BLAST, and therefore the fragments library may suffer.

Additionally, the Rosetta de novo protocol folds two divergent homologous sequences in addition to the query sequence. If PSI-BLAST cannot find any sequence homologs with confidence due to a query being exceptionally short, Rosetta cannot take advantage of multiple homologs, and the modeling may suffer.

Perhaps most importantly in de novo modeling, the assumption is made that the target protein forms a soluble domain with a hydrophobic core. Short sequences often do not fold up in this fashion, so the energy function used in Rosetta may incorrectly bias the structures to be more compact than they should be for short targets.

Question 18. What Is The Difference Between Ab Initio And De Novo Modeling?

Answer :

Ab initio structure prediction classically refers to structure prediction using nothing more than first-principles (i.e. physics). De Novo is a more general term that refers to the greater category of methods that do not use templates from homologous PDB structures. Since Rosetta uses fragments from existing PDB structures in order to guide the search in conjunction with energy functions, there is a semantic argument as to whether it is truly "ab initio" (although the same could be said for any statistically derived energy function). Long story short: call it what you want, but be prepared for a debate!

Question 19. What Is The Difference Between Comparative And Homology Modeling?

Answer :

Comparative vs. Homology Modeling: same thing, namely model a significant fraction of your target using coordinates from a homologous parent PDB structure.

Question 20. Does The Rosetta Software Come As Pre-compiled Executables, Or Source Code, In Which Language?

Answer :

We distribute source code in C++ and users need to compile it themselves.

Question 21. What's The Minimum And Recommended Hardware Settings For Rosetta Software?

Answer :

Rosetta requires a Unix-like operating system like Linux or MacOS - it will not easily run on a Windows machine.

To get the sampling needed for most Rosetta protocols, it's best to run Rosetta on a multiprocessor computing cluster. Most Rosetta protocols are trivially parallelizable, though, and can be run on a single processor, at the cost of much longer runtimes.

Most modern processors should be suitable. The one limitation you may run into is memory. We recommend at least 1G memory per CPU running Rosetta for best performance.

Question 22. What Is Rosie? Robetta?

Answer :

There are a number of publicly accessible servers on the web that allow researchers to run certain Rosetta protocols without installing Rosetta locally.

Robetta is the original Rosetta web server.

ROSIE (the Rosetta Online Server that Includes Everyone) is a new, centralized site for Rosetta web servers, and includes a number of protocols.

Other web servers also exist.

Question 23. Why Does Rosetta Delete Some Of My Residues?

Answer :

Rosetta will delete residues which are missing too many backbone atoms. "Missing" includes those atoms which are marked with zero occupancy in the PDB. Add the flag "-ignore_zero_occupancy false" to change this behavior.

If the backbone atoms are completely missing, use the loopmodel protocol to build in the residues.

Question 24. What Is Packing? What Is Design?

Answer :

Packing is the process of putting sidechains onto a backbone, or optimizing the conformation of sidechains. Rosetta uses a set of "rotamers" (discrete sidechain conformation samples derived from experimentally determined structures) and a Metropolis Monte Carlo simulated annealing process to determine which combination of rotamers produces the lowest energy for a given backbone.

Design in Rosetta uses the same packing machinery as sidechain optimization, but instead of optimizing within the rotamers for a single amino acid, it considers rotamers for a number of different amino acid identities.

Question 25. How Come Rosetta Is Reporting Rmsd To The Native Structure When There Is No Native Structure?

Answer :

Most protocols with use the input structure as a "mock native" structure.