title: X-ray sequence and crystal structure of luffaculin 1, a novel type 1 ribosome-inactivating protein.
abstract: BACKGROUND
  Protein sequence can be obtained through Edman degradation, mass spectrometry, or cDNA sequencing. High resolution X-ray crystallography can also be used to derive protein sequence information, but faces the difficulty in distinguishing the Asp/Asn, Glu/Gln, and Val/Thr pairs. Luffaculin 1 is a new type 1 ribosome-inactivating protein (RIP) isolated from the seeds of Luffa acutangula. Besides rRNA N-glycosidase activity, luffaculin 1 also demonstrates activities including inhibiting tumor cells' proliferation and inducing tumor cells' differentiation.
  
  
  RESULTS
  The crystal structure of luffaculin 1 was determined at 1.4 A resolution. Its amino-acid sequence was derived from this high resolution structure using the following criteria: 1) high resolution electron density; 2) comparison of electron density between two molecules that exist in the same crystal; 3) evaluation of the chemical environment of residues to break down the sequence assignment ambiguity in residue pairs Glu/Gln, Asp/Asn, and Val/Thr; 4) comparison with sequences of the homologous proteins. Using the criteria 1 and 2, 66% of the residues can be assigned. By incorporating with criterion 3, 86% of the residues were assigned, suggesting the effectiveness of chemical environment evaluation in breaking down residue ambiguity. In total, 94% of the luffaculin 1 sequence was assigned with high confidence using this improved X-ray sequencing strategy. Two N-acetylglucosamine moieties, linked respectively to the residues Asn77 and Asn84, can be identified in the structure. Residues Tyr70, Tyr110, Glu159 and Arg162 define the active site of luffaculin 1 as an RNA N-glycosidase.
  
  
  CONCLUSIONS
  X-ray sequencing method can be effective to derive sequence information of proteins. The evaluation of the chemical environment of residues is a useful method to break down the assignment ambiguity in Glu/Gln, Asp/Asn, and Val/Thr pairs. The sequence and the crystal structure confirm that luffaculin 1 is a new type 1 RIP.
authors.affiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, The Chinese Academy of Sciences, 155 Yang Qiao Xi Lu, Fuzhou, Fujian, China. houxiaomin@fjirsm.ac.cn <houxiaomin@fjirsm.ac.cn>
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Hou
authors.forename: Minghuang
authors.initials: M
authors.lastname: Chen
authors.forename: Liqing
authors.initials: L
authors.lastname: Chen
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Meehan
authors.forename: Jieming
authors.initials: J
authors.lastname: Xie
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
mesh_terms: D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D054789:Ribosome Inactivating Proteins, Type 1; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
chemical_list: D006023:Glycoproteins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C088243:luffaculin protein, Luffa acutangula