5. Input data description

5.1. Map

Modeling means atomic interpretation of a map. This map can be the result of our own reconstruction process or can be obtained from a database. In this tutorial we use the haemoglobin map EMD-3488, that can be downloaded from PDBe (Fig. 5.1).

WARNING In case you use your own map obtained from cryo-EM images: Take into account that cryo-EM 3D maps benefit significantly of an “optimizing” step, normally referred to as “sharpening” or “density improvement“, that tends to increase signal at medium/high resolution. Therefore, we recommend to sharp the map before tracing the atomic model. Either two Scipion protocols consecutively applied, xmipp3-local MonoRes [Vilas et al., 2018] and xmipp3-localdeblur sharpening [Ramírez-Aportela et al., 2018], or the protocol xmipp3-deepEMhancer [Sanchez-Garcia et al., 2020], allow map sharpening. Details about the parameters of these protocols are shown in Appendices Local MonoRes, Local Deblur Sharpening and DeepEMhancer Sharpening, respectively.

Fig. 5.1 Downloading the volume from PDBe.

Once downloaded the volume, unpack it (command line: gunzip emd-3488.map.gz) and save it in your tutorial folder.

5.2. Sequences

The sequences of Hgb \alpha and \beta subunits are included in UniProtKB. Accession numbers are P69905 and P68871, respectively. Next, we show both sequences in fasta format:

>sp|P69905|HBA_HUMAN Haemoglobin subunit alpha
MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHG
KKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTP
AVHASLDKFLASVSTVLTSKYR

>sp|P68871|HBB_HUMAN Haemoglobin subunit beta
MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPK
VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFG
KEFTPPVQAAYQKVVAGVANALAHKYH

These protein sequences were determined by direct translation from the experimental sequence obtained from complementary DNA (cDNA), i.e., DNA synthesized or retro-transcribed from messenger RNA (mRNA). In this way, it is quite unlikely that these sequences include post-translational modifications. Although methionine is added with the translation Met-tRNA initiation factor, the removal of methionine aminoacid from the N-terminus of a polypeptide is a common post-translational modification. Since Met appears at the N-terminal end of both proteins, we can predict that these are not the polypeptide mature forms and Met will be removed in the mature ones that are present in the atomic structures.

Those two sequences can be retrieved from UniProtKB using Scipion import sequence  protocol, which allows direct downloading from the database.