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BioSci M160 / MolBio255 Structure-Function Relationships of Integral Membrane Proteins Lecture 3 Hartmut “Hudel” Luecke Biochemistry, Biophysics & Computer Science

1.Wet lab tour (protein production, purification, crystallization) 2.X-ray lab virtual tour (data collection at the Stanford Synchrotron!) 3.3D structure visualization (programs Coot, Pymol etc.)

Without a crystal structure… Nucleotide sequence: GAGCCCACGAGAGGCAGCGCCATGGCGGAGCAGACCTACTCGTGGGCCTATTCCCTGGTGGATTCCAGTCAAGTGT CTACATTTCTGATTTCCATTCTTCTTATAGTCTATGGTAGTTTCAGGTCCCTTAATATGGACTTTGAAAATCAAGA TAAGGAGAAAGACAGTAATAGTTCTTCTGGGTCTTTCAATGGGGAACAGGAACCAATAATTGGCTTCCAACCAATG GACTCTACCCGGGCTCGGTTCCTTCCAATGGGAGCATGTGTCTCTCTTTTAGTAATGTTCTTCTTCTTTGACTCAG TTCAAGTAGTTTTTACAATATGTACAGCAGTTCTTGCAACGATAGCTTTTGCTTTTCTTCTCCTCCCGATGTGCCA GTATTTAACAAGACCCTGCTCACCTCAGAACAAGATTTCCTTTGGTTGCTGTGGACGTTTCACTGCTGCTGAGTTG CTGTCATTCTCTCTGTCTGTCATGCTCGTCCTCATCTGGGTTCTCACTGGCCATTGGCTTCTCATGGATGCACTGG CCATGGGCCTCTGTGTCGCCATGATCGCCTTTGTCCGCCTGCCGAGCCTCAAGGTCTCCTGCCTGCTTCTCTCAGG GCTTCTCATCTATGATGTCTTTTGGGTATTTTTCTCAGCCTACATCTTCAATAGCAACGTCATGGTGAAGGTGGCC ACTCAGCCGGCTGACAATCCCCTTGACGTTCTATCCCGGAAGCTCCACCTGGGGCCCAATGTTGGGCGTGATGTTC CTCGCCTGTCTCTGCCTGGAAAACTGGTCTTCCCAAGCTCCACTGGCAGCCACTTCTCCATGTTGGGCATCGGAGA CATCGTTATGCCTGGTCTCCTACTATGCTTTGTCCTTCGCTATGACAACTACAAAAAGCAAGCCAGTGGGGACTCC TGTGGGGCCCCTGGACCTGCCAACATCTCCGGGCGCATGCAGAAGGTCTCCTACTTTCACTGCACCCTCATCGGAT ACTTTGTAGGCCTGCTCACTGCTACTGTGGCGTCTCGCATTCACCGGGCCGCCCAGCCCGCCCTTCTCTATTTGGT GCCATTTACTTTATTGCCACTCCTCACGATGGCCTATTTAAAGGGCGACCTCCGGCGGATGTGGTCTGAGCCTTTC CACTCCAAGTCCAGCAGCTCCCGATTCCTGGAAGTATGATGGAT

Without a crystal structure… Amino acid sequence: MAEQTYSWAYSLVDSSQVSTFLISILLIVYGSFRSLNMDFENQDKEKDSNSSSGSFNGEQEPIIGFQPMDSTRARF LPMGACVSLLVMFFFFDSVQVVFTICTAVLATIAFAFLLLPMCQYLTRPCSPQNKISFGCCGRFTAAELLSFSLSV MLVLIWVLTGHWLLMDALAMGLCVAMIAFVRLPSLKVSCLLLSGLLIYDVFWVFFSAYIFNSNVMVKVATQPADNP LDVLSRKLHLGPNVGRDVPRLSLPGKLVFPSSTGSHFSMLGIGDIVMPGLLLCFVLRYDNYKKQASGDSCGAPGPA NISGRMQKVSYFHCTLIGYFVGLLTATVASRIHRAAQPALLYLVPFTLLPLLTMAYLKGDLRRMWSEPFHSKSSSS RFLEV

Without a crystal structure… Secondary structure prediction: Query_name: SSPL3 Query_length: 385 MAEQTYSWAYSLVDSSQVSTFLISILLIVYGSFRSLNMDFENQDKEKDSNSSSGSF NGEQ CCEEECCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCC CCCCCCCC EPIIGFQPMDSTRARFLPMGACVSLLVMFFFFDSVQVVFTICTAVLATIAFAFLLLPM CQ CCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHH YLTRPCSPQNKISFGCCGRFTAAELLSFSLSVMLVLIWVLTGHWLLMDALAMGLCV AMIA HCCCCCCCCCCCCEEECCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHH FVRLPSLKVSCLLLSGLLIYDVFWVFFSAYIFNSNVMVKVATQPADNPLDVLSRKLHL GP HHCCCCHHHHHHHHHHHHHHHHEHHEECHHHCCCCEEEEEECCCCCCCHHHH HHHCCCCC NVGRDVPRLSLPGKLVFPSSTGSHFSMLGIGDIVMPGLLLCFVLRYDNYKKQASGD SCGA CCEECCCCCCCCCCHCCCCCCCCCCEEECCCCEECHHHHHHEHEHHCHHHHH CCCCCCCC PGPANISGRMQKVSYFHCTLIGYFVGLLTATVASRIHRAAQPALLYLVPFTLLPLLTMA Y CCCHHHCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHCHHHHHHHHHH HHHHHHHHH LKGDLRRMWSEPFHSKSSSSRFLEV HHHHHHHHHCCCCCCCCCCCCEECC H = Helix E = Strand C = other

Without a crystal structure… Hydropathy plot using MPEx

Without a crystal structure… CASP!CASP

Wet lab (protein production, purification, crystallization) 1.Production: overexpression in E. coli, yeast, insect, CHO or human (HEK293) cellsProduction: overexpression in E. coli, yeast, insect, CHO or human (HEK293) cells 2.Extraction: detergent-extraction from membrane fraction after lysis of cells 3.Purification: chromatographic separation of detergent-solubilized protein (Ni column) 4.Concentration: increasing concentration to 2-20 mg / ml 5.Crystallization: vapor diffusion (sitting drop), cubic lipid phase (CLP) or bicelle setups

Membrane protein crystals

Membrane protein crystal diffraction 3 weeks5 months

X-ray lab: test diffraction quality of crystals Synchrotron: collect diffraction data How are synchrotron x- rays produced?

Why cryo (100 K nitrogen stream) data collection? Reduces radiation-induced decay

Why cryo data collection? Cryo-trapping of reaction intermediates

Why Cryo data collection? Cryo-trapping of reaction intermediates

Why Cryo data collection? Cryo-trapping of reaction intermediates

2 F(obs) – F(calc) electron density map 2 F(obs) – F(calc) = F(obs) + [F(obs) – F(calc)]

F(obs) – F(calc) electron density map (difference map)