How to predict structures with AlphaFold: Difference between revisions
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==Is An Empirical Model Available?== | ==Is An Empirical Model Available?== | ||
Empirical models are the most accurate, so you should look for those first. See [[How To Find A Structure]]. If there is no empirical model for your amino acid sequence, it may be useful to explore [[How_To_Find_A_Structure#Sequence-Related_Empirical_Models|empirical models for closely-related sequences]], if available. | Empirical models are the most accurate, so you should look for those first. See [[How To Find A Structure]]. If there is no empirical model for your amino acid sequence, it may be useful to explore [[How_To_Find_A_Structure#Sequence-Related_Empirical_Models|empirical models for closely-related sequences]], if available. Even if an empirical structure is available, most have missing residues or atoms, and it may be useful to compare it with the AlphaFold prediction: see [[Missing residues and incomplete sidechains]]. | ||
==Does AlphaFold Database Already Have Your Protein?== | ==Does AlphaFold Database Already Have Your Protein?== | ||
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==Prediction Servers== | ==Prediction Servers== | ||
This is not a comprehensive list. Please add other servers of interest to a broad range of users, including beginners. | You can submit one (or a set) of sequences to these servers, and they will return predicted structures, along with estimates of confidence in their predictions. This is not a comprehensive list. Please add other servers of interest to a broad range of users, including beginners. | ||
* | * 2024<ref name="af3" />: [https://alphafoldserver.com AlphaFoldServer.Com]. Using AlphaFold3, predicts homo- and hetro-multimers involving protein, DNA, RNA, ligands, and modified residues. Straightforward to use; Guide and FAQ provided. Predictions are templated without user control (see FAQ). Free for non-commercial use -- see [https://alphafoldserver.com/terms Terms of Service] and [https://alphafoldserver.com/output-terms Output Terms of Use]. From the DeepMind team<ref name="af3" />. | ||
**If you get <font color="red"><b>Invalid character</b></font> after pasting in your sequence, try removing the breaks between lines. | |||
**Predicted models are in [[Mmcif format|mmCIF format]] only. When visualized in [http://firstglance.jmol.org FirstGlance in Jmol], coloring by confidence/pLDDT is automatic. [[Converting AlphaFold3 CIF to PDB|More information about .cif and FirstGlance]]. | |||
**To easily obtain average [[pLDDT]] (predicted confidence) for a range of residues, see [[FirstGlance/How to get average pLDDT from AlphaFold models]]. | |||
**Chain ID assignments differ from those of empirical wwPDB files: see [[Chains_and_Chain_IDs#AlphaFold3_Chain_IDs|AlphaFold3 Chain IDs]]. | |||
**See also [[#Visualizing Predicted Structures]] and [[User:Eric Martz/AlphaFold3 case studies|AlphaFold3 case studies]]. | |||
* | * 2024<ref name="rfaa">PMID: 38452047</ref>: RosettaFold All-Atom (RFAA) predicts multimers of protein and nucleic acids with ligands. From the Baker team<ref name="rfaa" />. [https://neurosnap.ai/service/RoseTTAFold%20All-Atom A free server limited to very small numbers of jobs is available from Neurosnap]. | ||
* | * 2024 update? [https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb ColabFold AlphaFold2 and AlphaFold-multimer] predicts multiple chain complexes. Easy to use. From Mirdita ''et al.''<ref name="colabfold" /> | ||
* | * 2024<ref name="combfold">PMID: 38326495</ref>: [https://neurosnap.ai/service/CombFold CombFold] predicts the structures of large protein complexes from subunit sequences using AlphaFold Multimer paired with a combinatorial method to assemble subunits. From Shor and Schneidman-Duhovny<ref name="combfold" />. | ||
* | * 2022<ref name="colabfold">PMID: 35637307</ref>: [https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/AlphaFold2_advanced.ipynb ColabFold AlphaFold2_advanced]. Predicts homo- and hetero-multimers using methods from the Steinegger/Mirdita team<ref name="colabfold" /><ref name="colabfold2024">PMID: 39402428</ref>, before AlphaFold-multimer<ref name="afmultimer">[https://www.biorxiv.org/content/10.1101/2021.10.04.463034v1.full Protein complex prediction with AlphaFold-Multimer], Preprint, Evans et al. 2021.</ref> was available. Does NOT use templates. [https://www.ebi.ac.uk/training/online/courses/alphafold/accessing-and-predicting-protein-structures-with-alphafold/predicting-protein-structures-with-colabfold-and-alphafold-colab/ See Instructions from EMBL-EBI]. | ||
* | *2022<ref name="af2021">PMID: 34265844</ref><ref name="afmultimer" />: [https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb AlphaFold2/Multimer Colab] able to predict protein multimers. From the DeepMind team<ref name="af2021" /><ref name="afmultimer" />. [https://www.ebi.ac.uk/training/online/courses/alphafold/accessing-and-predicting-protein-structures-with-alphafold/predicting-protein-structures-with-colabfold-and-alphafold-colab/ See Instructions from EMBL-EBI]. | ||
=== | *2022<ref name="alphafill">PMID: 36424442</ref>: [https://alphafill.eu/ AlphaFill] “transplants” missing ligands, cofactors and (metal) ions into AlphaFold models. From the Perrakis team<ref name="alphafill" />. Ligand positioning is approximate. See [[Alphafold#Ligands:_AlphaFill |CAUTION]] provided by the AlphaFill team: | ||
<blockquote> | |||
"AlphaFill models are not meant or suitable for precise quantification of interactions between the transferred ligand(s) and the protein (e.g. hydrogen bonds, π-π or cation-π interactions, van der Waals interactions, hydrophobic interactions, halogen bonds)." | |||
</blockquote> | |||
*2021<ref name="rosettafold">PMID: 34282049</ref>: [https://robetta.bakerlab.org/ RoseTTAFold at Robetta] is an independent design from the Baker team<ref name="rosettafold" />, influenced by the design of AlphaFold2. Predicts monomers and multimers. Comparing results of RoseTTAFold with results of AlphaFold2/3 is worthwhile. At [https://robetta.bakerlab.org/ Robetta], open the Structure Prediction menu at the top, and choose Submit. ''Be sure to check RoseTTAFold under Optional!'' | |||
===Cost?=== | |||
The above servers are free for limited, non-commercial use. | The above servers are free for limited, non-commercial use. | ||
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[http://firstglance.jmol.org FirstGlance in Jmol] automatically colors its initial view of | [http://firstglance.jmol.org FirstGlance in Jmol] automatically colors its initial view of AlphaFold or RoseTTAFold models by estimated confidence [[pLDDT]] ('''{{Font color|blue|blue for high confidence}}, {{Font color|red|red for low confidence}}'''). After you go to other views or tools, you can always get back to this color scheme by clicking ''Reliability Estimates'' in the ''Views'' tab. | ||
*[[iCn3D]] automatically colors AlphaFold2 Database models loaded from their UniProt IDs. For AlphaFold files opened from your computer, use pLDDT on the pull-down Color menu. | |||
*[[PyMOL]] and [[ChimeraX]] have no built-in confidence/pLDDT color scheme. Their rainbow/spectrum color schemes for temperature/B-factor color confidence/pLDDT with the AlphaFold color scheme inverted. | |||
[http://firstglance.jmol.org/where.htm#uploading Upload] your predicted PDB file to [http://firstglance.jmol.org FirstGlance.Jmol.Org], which has many [http://firstglance.jmol.org/whatis.htm#unique unique conveniences and capabilities]. | [http://firstglance.jmol.org/where.htm#uploading Upload] your predicted PDB file to [http://firstglance.jmol.org FirstGlance.Jmol.Org], which has many [http://firstglance.jmol.org/whatis.htm#unique unique conveniences and capabilities]. | ||
When using [http://FirstGlance.jmol.org FirstGlance], it is easy to visualize | |||
* Estimated reliability | * Estimated confidence/pLDDT by touching an atom | ||
* '''Average confidence/pLDDT''' ("reliability") for the entire model, or for [[FirstGlance/How to get average pLDDT from AlphaFold models|a specified sequence range]]. | |||
* Secondary structure (Views tab) | * Secondary structure (Views tab) | ||
* Distribution of hydrophobic vs. polar residues (Views tab: integral membrane proteins will have large hydrophobic surfaces while soluble proteins will have hydrophobic cores revealed by the ''Slab'' button) | * Distribution of hydrophobic vs. polar residues (Views tab: integral membrane proteins will have large hydrophobic surfaces while soluble proteins will have hydrophobic cores revealed by the ''Slab'' button) | ||
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</ref>. In 2024, ColabFold is not necessarily the best or only place to submit you job: see [[#Prediction Servers]]. | </ref>. In 2024, ColabFold is not necessarily the best or only place to submit you job: see [[#Prediction Servers]]. | ||
Initially, AlphaFold and ColabFold performed best with '''single chains'''<ref name=" | Initially, AlphaFold and ColabFold performed best with '''single chains'''<ref name="afmultimer">[https://www.biorxiv.org/content/10.1101/2021.10.04.463034v1 Protein complex prediction with AlphaFold-Multimer], 2021, Evans ''et al.'' (DeepMind Team).</ref>, which may include one or a few domains. The instructions below were written '''before ColabFold was adapted to prediction of multimers'''. If you are interested in complexes or alternate conformations, please see ColabFold instructions in the 2023 paper by Kim ''et al.'' <ref name="kim2023">[https://protocolexchange.researchsquare.com/article/pex-2490/v1 Easy and accurate protein structure prediction using ColabFold], 2023, Kim ''et al.'' (DeepMind Team).</ref> | ||
===Submitting A Sequence=== | ===Submitting A Sequence=== | ||
First, check the [https://alphafold.ebi.ac.uk/ AlphaFold Database] for the protein of interest. If its structure has already been predicted there, download it, and skip to [[#Interpreting Results|Interpreting Results]] below. Otherwise ... | First, if your query is a single chain molecule, check the [https://alphafold.ebi.ac.uk/ AlphaFold Database] for the protein of interest. If its structure has already been predicted there, download it, and skip to [[#Interpreting Results|Interpreting Results]] below. Otherwise ... | ||
Don't worry about any of the options not specifically mentioned below. Leave them at their default settings. | Don't worry about any of the options not specifically mentioned below. Leave them at their default settings. | ||
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Each residue has an estimated reliability of its position (0-100) in the PDB [[temperature]] column. BEWARE that high values mean high confidence, and low values mean low confidence. This is the INVERSE of [[temperature|crystallographic temperature values]], where low values are good and high values are bad. Uploading your PDB file to [http://firstglance.jmol.org FirstGlance in Jmol] will automatically color each residue by its estimated reliability. | Each residue has an estimated reliability of its position (0-100) in the PDB [[temperature]] column. BEWARE that high values mean high confidence, and low values mean low confidence. This is the INVERSE of [[temperature|crystallographic temperature values]], where low values are good and high values are bad. Uploading your PDB file to [http://firstglance.jmol.org FirstGlance in Jmol] will automatically color each residue by its estimated reliability. | ||
====Intrinsic Disorder==== | ====Intrinsic Disorder==== | ||
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==See Also== | ==See Also== | ||
*[[AlphaFold/Index]], a list of pages in Proteopedia about Alphafold. | *[[AlphaFold/Index]], a list of pages in Proteopedia about Alphafold. | ||
*[[User:Eric Martz/AlphaFold3 case studies|AlphaFold3 case studies]] includes a case that AlphaFold3 cannot predict. | |||
*[[How To Find A Structure]] | |||
*[[Missing residues and incomplete sidechains]] | |||
==References and Notes== | ==References and Notes== | ||
<references /> | <references /> | ||
