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Model building and refinement

QS Qiang Su
YS Yigong Shi

Last updated date: Dec 25, 2022 Views: 704 Forks: 0

original An abbreviated version of this protocol was published in Science
Cryo-EM structure of the human IgM B cell receptor
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The sequence used for model building is the mIgM antibody.

 

The full-length mIgM sequence is listed below:

 

Heavy Chain:

MEFGLSWLFLVAILKGVQCQVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTEGEVSADEEGFENLWATASTFIVLFLLSLFYSTTVTLFKVK

 

The heavy chain comprises three parts: synthetic signal peptide (10.1371/journal.pone.0116878), variable domain of VRC0 (10.1126/science.1192819), and constant domain of mIgM (Uniprot ID: P01871-2, https://rest.uniprot.org/uniprotkb/P01871-2.fasta). The three parts are highlighted by yellow, green and magenta color, respectively.

The codon optimized cDNA sequence:

ATGGAGTTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGGGTGCAGTGCCAAGTGCAGCTGGTGCAGAGCGGCGGGCAGATGAAGAAGCCCGGCGAGAGCATGAGAATCAGCTGCAGAGCTAGCGGCTACGAGTTCATCGACTGCACCCTGAACTGGATCAGACTGGCCCCCGGCAAGAGACCCGAGTGGATGGGCTGGCTGAAGCCTAGAGGCGGCGCCGTGAACTACGCTAGACCCCTGCAAGGCAGAGTGACCATGACAAGAGACGTGTACAGCGACACCGCCTTCCTGGAGCTGAGAAGCCTGACCGTGGACGACACCGCCGTGTACTTCTGCACAAGAGGCAAGAACTGCGACTACAACTGGGACTTCGAACACTGGGGGAGAGGCACCCCCGTGATCGTGAGCAGCGGCAGCGCTTCCGCCCCCACCCTGTTCCCCCTGGTGAGCTGCGAGAACAGCCCTAGCGATACAAGCAGCGTGGCCGTGGGCTGCCTGGCCCAAGACTTCCTGCCCGACAGCATCACCTTCAGCTGGAAGTACAAGAACAACAGCGACATCAGCAGCACAAGAGGCTTCCCTAGCGTGCTGAGAGGCGGCAAGTACGCCGCCACAAGCCAAGTGCTGCTGCCTAGCAAGGACGTGATGCAAGGCACCGACGAGCACGTGGTGTGCAAGGTGCAGCACCCCAACGGCAACAAGGAGAAGAACGTGCCCCTGCCCGTGATCGCCGAGCTGCCCCCCAAGGTGAGCGTGTTCGTGCCCCCTAGAGACGGCTTCTTCGGCAACCCTAGAAAGAGCAAGCTGATCTGCCAAGCCACCGGCTTCAGCCCCCGGCAGATCCAAGTGAGCTGGCTGAGAGAGGGCAAGCAAGTGGGCTCCGGCGTGACAACCGACCAAGTGCAAGCCGAGGCCAAGGAGAGCGGCCCCACCACCTACAAGGTGACAAGCACCCTGACCATCAAGGAGAGCGACTGGCTGGGGCAGAGCATGTTCACCTGCAGAGTGGACCACAGAGGCCTGACCTTTCAGCAGAACGCTAGCAGCATGTGCGTGCCCGACCAAGACACCGCCATCAGAGTGTTCGCCATCCCCCCTAGCTTCGCTAGCATCTTCCTGACCAAGAGCACCAAGCTGACCTGCCTCGTGACCGATCTGACCACCTACGACAGCGTGACCATCAGCTGGACAAGACAGAACGGCGAGGCCGTGAAGACCCACACCAACATCAGCGAGAGCCACCCCAACGCCACCTTCAGCGCCGTGGGCGAGGCTAGCATCTGCGAGGACGACTGGAACAGCGGCGAGAGATTCACCTGCACCGTGACCCACACCGACCTGCCTAGCCCCCTGAAGCAGACCATCAGCAGACCCAAGGGCGTGGCCCTGCACAGACCCGACGTGTACCTGCTGCCCCCCGCTAGAGAGCAGCTGAACCTGAGAGAGAGCGCCACCATCACCTGCCTGGTGACCGGCTTCAGCCCCGCCGATGTGTTCGTGCAGTGGATGCAGAGAGGGCAGCCCCTGAGCCCCGAGAAGTACGTGACAAGCGCTCCCATGCCTGAGCCCCAAGCCCCCGGCAGATACTTCGCCCACAGCATCCTGACCGTGAGCGAGGAAGAGTGGAACACCGGCGAGACCTACACCTGCGTGGTGGCCCACGAGGCCCTGCCCAACAGAGTGACCGAGAGAACCGTGGACAAGAGCACCGAGGGCGAGGTGAGCGCCGACGAGGAGGGCTTCGAGAACCTGTGGGCCACCGCTAGCACCTTCATCGTGCTGTTCCTGCTGAGCCTGTTCTACAGCACCACCGTGACCCTGTTCAAGGTGAAGTGA

 

Light Chain:

MVLQTQVFISLLLWISGAYGGSDYKDDDDKGSPGDEVDAGEIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

 

 

The codon optimized cDNA sequence:

ATGGTGCTGCAGACCCAAGTGTTCATCAGCCTGCTCCTGTGGATCAGCGGCGCCTACGGCGGCAGCGACTACAAAGATGACGATGACAAGGGCAGCCCCGGCGACGAGGTGGATGCCGGCGAGATCGTGCTGACACAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGACCGCCATCATCAGCTGCAGAACAAGCCAATACGGCAGCCTGGCCTGGTATCAGCAGAGACCCGGCCAAGCCCCTAGACTGGTGATCTACAGCGGCAGCACAAGAGCCGCCGGCATCCCCGACAGATTCAGCGGCAGCAGATGGGGCCCCGACTACAACCTGACCATCAGCAACCTGGAGAGCGGCGACTTCGGCGTGTACTACTGTCAGCAGTACGAGTTCTTCGGCCAAGGCACCAAGGTGCAAGTGGACATCAAGAGAACCGTGGCCGCCCCTAGCGTGTTCATCTTCCCCCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAAGAGAGCGTGACCGAGCAAGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAAGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGAGGCGAGTGCTGA

The light chain comprises five parts: native signal peptide (Human Ig kappa chain V-IV region B17, https://doi.org/10.1038/nprot.2017.126), flag tag flanked by GS linkers, DrICE cutting site (10.1038/nsmb764), variable domain of VRC0 (10.1126/science.1192819), and constant domain of Igκ(Uniprot ID: P01834-1, https://rest.uniprot.org/uniprotkb/P01834.fasta). The five parts are highlighted by yellow, blue, red, green and magenta color, respectively.

 

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Su, Q and Shi, Y(2022). Model building and refinement. Bio-protocol Preprint. bio-protocol.org/prep2094.
  2. Su, Q., Chen, M., Shi, Y., Zhang, X., Huang, G., Huang, B., Liu, D., Liu, Z. and Shi, Y.(2022). Cryo-EM structure of the human IgM B cell receptor. Science 377(6608). DOI: 10.1126/science.abo3923

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