Information

NAG, FUC molecules in PDB files


In some proteins (such as 4ZXB, 6CE9 which are respectively apo and halo forms of insulin receptors), I see ligands such as FUC (ALPHA-L-FUCOSE) and NAG(N-Acetylglucosamine). No matter which paper I checked I see no explanation for these ligands and therefore I assume they have to do with the experimental procedure but I do not quite understand what is their role. Do they stabilize a particular state of the protein? Note that the proteins above are determined by different methods the first one X-ray crystallography and the second by electron microscopy. I have no technical knowledge about the neither method so any input or link to a paper that actually explains them would be appreciated.

Thanks


The insulin receptor is a glycoprotein. This is discussed, albeit briefly, in both papers for the structures you mentioned and the glycosylation is visible in the structures themselves. You can get more info in the following paper, among others:

Sparrow LG, Lawrence MC, Gorman JJ, Strike PM, Robinson CP, McKern NM, Ward CW. 2008. N-linked glycans of the human insulin receptor and their distribution over the crystal structure. Proteins 71(1):426-439.


Apologia

This addresses the general case, rather than the particular case of the insulin receptor, which is answered by @canadianer.

The General Problem of unexpected ligands in PDB structures

In preparing a relation database of about 400 protein structures I encountered the problem of distinguishing ligands that were substrates or cofactors of the enzyme from those that were not. And, believe me, the latter were widespread (and a real pain). In some cases it was obvious that the 'ligand' was the buffer (often MES) in which the protein was dissolved or ions such as sulphate. In the case of sugars, I assumed that the latter were present to aid crystalization. That view seems to be supported - in some cases at least - by a review by McPherson and Gavira. In a list of eight categories of additives that are used in protein crystallization they include:

(v) Osmolytes, co-solvents and cosmotropes are compounds that exert their effects at relatively high concentrations, 1 M or more, and include a wide range of molecules such as sucrose, trehalose and other sugars, proline, TMAO, glycine, betaine, taurine, sarcosine and a host of others. The effect of their inclusion in the mother liquor is to stabilize (or destabilize) the native conformation of the protein by altering the interaction of the surface of the protein with water, or by altering the hydration layer and possibly the structured waters.

Example

As an example I cite the seven molecules of glycerol in 1B6G (shown below), a haloalkane dehalogenase from the bacterium, Xanthobacter autotrophicus. Although certain, usually pathogenic, bacteria do have glycosylation pathways, there is no suggestion that this is the case here, as indicated by the nature of the triose and the fact that the crystallization was performed in a glycerol solution:

Subsequently, the crystal was equilibrated for 0.5 h in a solution containing 70% ammonium sulfate and 100 mM MES buffer pH 5.0 and was then soaked for 3 h at room tempera- ture in 10 mM 1-chloropentane, 70% ammonium sulfate and 100 mM citrate buffer pH 5.0. A solution of 30%(w/v) PEG 6000, 20%(v/v) glycerol and 100 mM citrate pH 5.0 was applied as cryoprotectant during data collection.

Haloalkane dehalogenase, 1B6G. The glycerol molecules (grey and red) are shown in space-filling mode. (The yellow and red molecule is sulphate, the green one a chloride ion.)


Heterogen Section (updated)

The heterogen section of a PDB formatted file contains the complete description of non-standard residues in the entry. Detailed chemical definitions of non-polymer chemical components are described in the Chemical Component Dictionary (https://ftp.wwpdb.org/pub/pdb/data/monomers)

HET records are used to describe non-standard residues, such as prosthetic groups, inhibitors, solvent molecules, and ions for which coordinates are supplied. Groups are considered HET if they are not part of a biological polymer described in SEQRES and considered to be a molecule bound to the polymer, or they are a chemical species that constitute part of a biological polymer and is not one of the following:

  • standard amino acids, or
  • standard nucleic acids (C, G, A, U, I, DC, DG, DA, DU, DT and DI), or
  • unknown amino acid (UNK) or nucleic acid (N) where UNK and N are used to indicate the unknown residue name.

HET records also describe chemical components for which the chemical identity is unknown, in which case the group is assigned the hetID UNL (Unknown Ligand).

The heterogen section of a PDB formatted file contains the complete description of non-standard residues in the entry.

Record Format

  • Each HET group is assigned a hetID of not more than three (3) alphanumeric characters. The sequence number, chain identifier, insertion code, and number of coordinate records are given for each occurrence of the HET group in the entry. The chemical name of the HET group is given in the HETNAM record and synonyms for the chemical name are given in the HETSYN records, see https://ftp.wwpdb.org/pub/pdb/data/monomers .
  • There is a separate HET record for each occurrence of the HET group in an entry.
  • A particular HET group is represented in the PDB archive with a unique hetID.
  • PDB entries do not have HET records for water molecules, deuterated water, or methanol (when used as solvent).
  • Unknown atoms or ions will be represented as UNX with the chemical formula X1. Unknown ligands are UNL unknown amino acids are UNK.

Verification/Validation/Value Authority Control

For each het group that appears in the entry, the wwPDB checks that the corresponding HET, HETNAM, HETSYN, FORMUL, HETATM, and CONECT records appear, if applicable. The HET record is generated automatically using the Chemical Component Dictionary and information from the HETATM records.

Each unique hetID represents a unique molecule.

Relationships to Other Record Types

For each het group that appears in the entry, there must be corresponding HET, HETNAM, HETSYN, FORMUL,HETATM, and CONECT records. LINK records may also be created.


NAG, FUC molecules in PDB files - Biology

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.26 Å
  • R-Value Free: 0.237 
  • R-Value Work: 0.192 
  • R-Value Observed: 0.194 

wwPDB Validation   3D Report Full Report

Antibody Fucosylation Lowers the Fc gamma RIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan.

(2018) ACS Chem Biol 13: 2179-2189

  • PubMed: 30016589  Search on PubMedSearch on PubMed Central
  • DOI: 10.1021/acschembio.8b00342
  • Primary Citation of Related Structures:  
    5VU0
  • PubMed Abstract: 

Therapeutic monoclonal antibodies (mAbs) are largely based on the immunoglobulin G1 (IgG1) scaffold, and many elicit a cytotoxic cell-mediated response by binding Fc γ receptors. Core fucosylation, a prevalent modification to the asparagine (N)-linked carbohydrate on the IgG1 crystallizable fragment (Fc), decreases the Fc γ receptor IIIa (CD16a) binding affinity and mAb efficacy .

Therapeutic monoclonal antibodies (mAbs) are largely based on the immunoglobulin G1 (IgG1) scaffold, and many elicit a cytotoxic cell-mediated response by binding Fc γ receptors. Core fucosylation, a prevalent modification to the asparagine (N)-linked carbohydrate on the IgG1 crystallizable fragment (Fc), decreases the Fc γ receptor IIIa (CD16a) binding affinity and mAb efficacy. We determined IgG1 Fc fucosylation reduced the CD16a affinity by 1.7 ± 0.1 kcal/mol when compared to that of afucosylated IgG1 Fc however, CD16a N-glycan truncation decreased this penalty by 1.2 ± 0.1 kcal/mol or 70%. Fc fucosylation restricted the manifold of conformations sampled by displacing the CD16a Asn162-glycan that impinges upon the linkage between the α-mannose(1-6)β-mannose residues and promoted contacts with the IgG Tyr296 residue. Fucosylation also impacted the IgG1 Fc structure as indicated by changes in resonance frequencies and nuclear spin relaxation observed by solution nuclear magnetic resonance spectroscopy. The effects of fucosylation on IgG1 Fc may account for the remaining 0.5 ± 0.1 kcal/mol penalty of fucosylated IgG1 Fc binding CD16a when compared to that of afucosylated IgG1 Fc. Our results indicated the CD16a Asn162-glycan modulates the antibody affinity indirectly by reducing the volume sampled, as opposed to a direct mechanism with intermolecular glycan-glycan contacts previously proposed to stabilize this system. Thus, antibody engineering to enhance intermolecular glycan-glycan contacts will likely provide limited improvement, and future designs should maximize the affinity by maintaining the CD16a Asn162-glycan conformational heterogeneity.

Organizational Affiliation

Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology , Iowa State University , 2437 Pammel Drive, Molecular Biology Building, Room 4210 , Ames , Iowa 50011 , United States.


NAG, FUC molecules in PDB files - Biology

The PDB gives no guidelines for the use of chain identifiers, other than that one is encouraged to use certain characters:

However, a few rules-of-thumb have emerged over the years:

  1. If it is one chain in biology it should have one chain-id in the PDB file
  2. Chain-ids are preferably given as either logical abbreviations (E and I for Enzyme and Inhibitor or L and H for Light and Heavy chain), or in increasing alphabetical order (A, B, C, D, etc).
  3. If there is no need for the use of chain identifiers it was habit in the past not to use any. Lately, the chain-id A is used for such cases.

For the latter it is a pity that abc. xyz come before ABC. XYZ in the ASCII code listed above. Because we normally start with the capitals and use numericals and lower case characters "in case of emergency" only, which for the lower case characters breaks rule of thumb number 2.

EU name: 1K7C

In the file 1k7c.pdb the protein has chain-id A one of the sugars too. Most other sugars have chain-id B, but the sulphates and the waters have no chain-id at all.

Additionally, it is somewhat funny to see that the sugars are fully bound by symmetry related molecules. Normally the presence of sugars make crystallisation difficult, but in this case, it seems the sugars were important for forming the crystals.

The structure of 1k7c. The very thin lines represent a layer of 10 Ångström of residues (etcetera) in symmetry related molecules. The waters were not shown for clarity.

The same situation, but now rotating to see things better.

The structure again. The red NAG has chain-id A, just like the protein it is bound to. The yellow NAG, also bound to the protein, has chain-id B.

1E4M 1MYP

In 1e4m everything (amino acids, PO 4 , Zn, has chain-id M, but the waters have chain-id X. In 1MYP the sugars have a different chain-id from the residues they are covalently bound to.

EU name: 1HTR

This file contains one very intruiging water.

In 1htr none of the water molecules has a chain-id, except for one that has chain-id B. Indicated as a red-dotted yellow sphere.

And here the structure is rotating around. No matter how I look at it the water with chain-id B remains an intruiging mistery.


NAG, FUC molecules in PDB files - Biology

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.60 Å
  • R-Value Free: 0.252 
  • R-Value Work: 0.181 
  • R-Value Observed: 0.184 

wwPDB Validation   3D Report Full Report

Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus.

(2010) Science 328: 357-360

  • PubMed: 20339031  Search on PubMedSearch on PubMed Central
  • DOI: 10.1126/science.1186430
  • Primary Citation of Related Structures:  
    3LZF, 3LZG
  • PubMed Abstract: 

The 2009 H1N1 swine flu is the first influenza pandemic in decades. The crystal structure of the hemagglutinin from the A/California/04/2009 H1N1 virus shows that its antigenic structure, particularly within the Sa antigenic site, is extremely similar to those of human H1N1 viruses circulating early in the 20th century .

The 2009 H1N1 swine flu is the first influenza pandemic in decades. The crystal structure of the hemagglutinin from the A/California/04/2009 H1N1 virus shows that its antigenic structure, particularly within the Sa antigenic site, is extremely similar to those of human H1N1 viruses circulating early in the 20th century. The cocrystal structure of the 1918 hemagglutinin with 2D1, an antibody from a survivor of the 1918 Spanish flu that neutralizes both 1918 and 2009 H1N1 viruses, reveals an epitope that is conserved in both pandemic viruses. Thus, antigenic similarity between the 2009 and 1918-like viruses provides an explanation for the age-related immunity to the current influenza pandemic.

Organizational Affiliation

Department of Molecular Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.


NAG, FUC molecules in PDB files - Biology

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.70 Å
  • R-Value Free: 0.283 
  • R-Value Work: 0.217 
  • R-Value Observed: 0.220 

wwPDB Validation   3D Report Full Report

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains.

(2019) J Mol Biol 431: 3740-3752

  • PubMed: 31310764  Search on PubMedSearch on PubMed Central
  • DOI: 10.1016/j.jmb.2019.07.018
  • Primary Citation of Related Structures:  
    6RBF
  • PubMed Abstract: 

The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion .

The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion. Important open questions are how multiple mucin 2 dimers become covalently linked to one another and how mucin 2 multimerization compares with analogous processes in related polymers such as respiratory tract mucins and the hemostasis protein von Willebrand factor. Here we report the x-ray crystal structure of the mucin 2 multimerization module, found to form a dimer linked by two intersubunit disulfide bonds. The dimer structure calls into question the current model for intestinal mucin assembly, which proposes disulfide-mediated trimerization of the same module. Key residues making interactions across the dimer interface are highly conserved in intestinal mucin orthologs, supporting the physiological relevance of the observed quaternary structure. With knowledge of the interface residues, it can be demonstrated that many of these amino acids are also present in other mucins and in von Willebrand factor, further indicating that the stable dimer arrangement reported herein is likely to be shared across this functionally broad protein family. The mucin 2 module structure thus reveals the manner by which both mucins and von Willebrand factor polymerize, drawing deep structural parallels between macromolecular assemblies critical to mucosal epithelia and the vasculature.


NAG, FUC molecules in PDB files - Biology

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.70 Å
  • R-Value Free: 0.287 
  • R-Value Work: 0.231 
  • R-Value Observed: 0.231 

wwPDB Validation   3D Report Full Report

Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling.

(2017) Sci Rep 7: 37716-37716

  • PubMed: 28134246  Search on PubMedSearch on PubMed Central
  • DOI: 10.1038/srep37716
  • Primary Citation of Related Structures:  
    5FUC
  • PubMed Abstract: 

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the "junctional epitope" nature of VHH6, a camelid single domain antibody recognizing the IL-6-gp80 complex but not the individual components alone .

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the "junctional epitope" nature of VHH6, a camelid single domain antibody recognizing the IL-6-gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions.


NAG, FUC molecules in PDB files - Biology

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.66 Å
  • R-Value Free: 0.272 
  • R-Value Work: 0.244 
  • R-Value Observed: 0.245 

wwPDB Validation   3D Report Full Report

Structural basis of toll-like receptor 3 signaling with double-stranded RNA.

(2008) Science 320: 379-381

  • PubMed: 18420935  Search on PubMedSearch on PubMed Central
  • DOI: 10.1126/science.1155406
  • Primary Citation of Related Structures:  
    3CIG, 3CIY
  • PubMed Abstract: 

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction .

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.

Organizational Affiliation

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.


Biological Assemblies Related

Issue: struct_asym_id in assembly/ in_chains generate new struct_asym_id

The in_chains column contains a new version of struct_asym_id for that particular assemble and entity. The rule seems to be like this: In a specified assemble, for those original chains, keep their original struct_asym_id for those replicated chains, generate new struct_asym_id that continues the original chain’s struct_asym_id while not in collision with existing struct_asym_id in that assemble (i.e. assembly3’s <entity3’s PB PC><entity5’s PA>, assembly2’s <entity3’s PA PC><entity5’s PB>).

And I found that the ID of chain provided by PDBe PISA API (i.e. https://www.ebi.ac.uk/pdbe/api/pisa/interfacelist/2o2q/3) are corresponding to the above ID (at least in my usage cases):

Since I can get all the struct_asym_id in the asymmetric unit and their replication result from mmCIF’s _pdbx_struct_assembly_gen and _pdbx_struct_oper_list and I can infer their model_id and ranking:

I would like to apply the automatic rule to generate corresponding new struct_asym_id for a specified assemble and map them with the interfacelist provided by PISA API.

The issue is, what is the automatic rule?

Related Issues

    : Better support for symmetry in the Structure model : Biological assembly expansion: chain ids should contain both operator ids in binary expression case
      : Assembly chain ids for cases with composed operators in assembly expansion

    Carbohydrate 3D structure validation

    Typical problems of carbohydrate moieties in wwPDB entries are illustrated.

    Validation tools to identify these issues are described.

    Recommendations how to reduce the number of new issues are given.

    Approaches to rectify incorrect glycan 3D-structures are shown.

    Glycoproteins and protein–carbohydrate complexes in the worldwide Protein Data Bank (wwPDB) can be an excellent source of information for glycoscientists. Unfortunately, a rather large number of errors and inconsistencies is found in the glycan moieties of these 3D structures. This review illustrates frequent problems of carbohydrate moieties in wwPDB entries, such as nomenclature issues, incorrect N-glycan core structures, missing or erroneous linkages, or poor glycan geometry, and describes the carbohydrate-specific validation tools that are designed to identify such problems. Recommendations how to avoid these issues or how to rectify incorrect structures are also given.


    Watch the video: Why People Risk Their Lives To Bleach Their Skin. Shady. Refinery29 (November 2021).