Document Detail

Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis.
Jump to Full Text
MedLine Citation:
PMID:  22622584     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence and development. CHI operates near the diffusion limit with stereospecific control. Although associated primarily with plants, the CHI fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically 'perfected' enzyme from a non-enzymatic ancestor over a defined period of plant evolution.
Authors:
Micheline N Ngaki; Gordon V Louie; Ryan N Philippe; Gerard Manning; Florence Pojer; Marianne E Bowman; Ling Li; Elise Larsen; Eve Syrkin Wurtele; Joseph P Noel
Related Documents :
17053274 - Rapid characterization of the fatty acyl composition of complex lipids by collision-ind...
21414764 - Glycyrrhizin and glycyrrhetinic acid directly modulate rat cardiac performance.
2278824 - Ceroid accumulation by murine peritoneal macrophages exposed to artificial lipid-contai...
2230224 - Unusual cholesterol esters in the sebum of young children.
6999804 - Rapid micromethods for detecting deamination and decarboxylation of amino acids, indole...
697354 - Kinetic parameters and relative turnovers of some important catabolic reactions in dige...
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2012-05-13
Journal Detail:
Title:  Nature     Volume:  485     ISSN:  1476-4687     ISO Abbreviation:  Nature     Publication Date:  2012 May 
Date Detail:
Created Date:  2012-05-24     Completed Date:  2012-07-17     Revised Date:  2014-01-09    
Medline Journal Info:
Nlm Unique ID:  0410462     Medline TA:  Nature     Country:  England    
Other Details:
Languages:  eng     Pagination:  530-3     Citation Subset:  IM    
Data Bank Information
Bank Name/Acc. No.:
PDB/4DOI;  4DOK;  4DOL;  4DOO
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Arabidopsis / chemistry*,  enzymology,  genetics,  growth & development
Arabidopsis Proteins / chemistry,  genetics,  metabolism
Biocatalysis*
Crystallography, X-Ray
Evolution, Molecular*
Fatty Acid-Binding Proteins / chemistry,  deficiency,  genetics,  metabolism
Fatty Acids / metabolism*
Intramolecular Lyases / chemistry*,  deficiency,  genetics,  metabolism*
Ligands
Models, Molecular
Phenotype
Protein Binding
Protein Folding*
Stereoisomerism
alpha-Linolenic Acid / metabolism
Grant Support
ID/Acronym/Agency:
CA14195/CA/NCI NIH HHS; //Howard Hughes Medical Institute; //Howard Hughes Medical Institute
Chemical
Reg. No./Substance:
0/Arabidopsis Proteins; 0/Fatty Acid-Binding Proteins; 0/Fatty Acids; 0/Ligands; 0RBV727H71/alpha-Linolenic Acid; EC 5.5.-/Intramolecular Lyases; EC 5.5.1.6/chalcone isomerase
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-journal-id): 0410462
Journal ID (pubmed-jr-id): 6011
Journal ID (nlm-ta): Nature
Journal ID (iso-abbrev): Nature
ISSN: 0028-0836
ISSN: 1476-4687
Article Information
Download PDF

License:
nihms-submitted publication date: Day: 3 Month: 4 Year: 2012
Electronic publication date: Day: 13 Month: 5 Year: 2012
pmc-release publication date: Day: 04 Month: 1 Year: 2014
Volume: 485 Issue: 7399
E-location ID: 10.1038/nature11009
PubMed Id: 22622584
ID: 3880581
DOI: 10.1038/nature11009
ID: HHMIMS360927

Evolution of the Chalcone Isomerase Fold from Fatty Acid-Binding to Stereospecific Enzyme
Micheline N. Ngaki1*
Gordon V. Louie2*
Ryan N. Philippe2*
Gerard Manning3
Florence Pojer2
Marianne E. Bowman2
Ling Li1
Elise Larsen2
Eve Syrkin Wurtele1
Joseph P. Noel2
1Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011, USA
2Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology and Proteomics
3Razavi Newman Center for Bioinformatics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA
Correspondence: Correspondence to: Eve Syrkin Wurtele1 or Joseph P. Noel2 Correspondence and requests for materials should be addressed to E.S.W. (mash@iastate.edu) or J.P.N. (noel@salk.edu
*These authors contributed equally to this manuscript.

CHI (EC 5.5.1.6) catalyzes the intramolecular and stereospecific cyclization of chalcones to chiral flavanones via a Michael addition reaction7. The origin of CHI has been a mystery, due to the apparent absence of a related protein from primary metabolism8. CHI-like homologues in fungi and bacteria9 lack both key catalytic residues and the chalcone-binding site of bona fide CHI (Fig. 1a-c, Supplementary Fig. 1). Phylogenetic analysis shows that CHI is restricted to vascular plants, and is derived from FAP3. FAP3 forms one of two branches of FAP proteins (FAP1 together with FAP2 form the other) found in derived and basal plants and is likely homologous to CHI-fold proteins from protists and fungi (Supplementary Figs. 2,3, Supplementary Files 1,2). A. thaliana has five genes (and one pseudogene not shown) encoding CHI-fold proteins: two CHI/CHI-like (CHIL) members, AtCHI (At3g55120) and AtCHIL (At5g05270), and three FAP members, AtFAP1, AtFAP2 and AtFAP3 (At3g63170, At2g26310, and At1g53520, respectively). Pairwise sequence identity of the shared CHI-fold domain ranges from 10% to 63%. AtCHI is a bona fide enzyme, and mutations of its gene result in plants devoid of flavonoids 10. The active site of AtCHI retains residues important for catalytic activity, and AtCHI catalyzes the in vitro formation of (2S)-naringenin (Fig. 1c). In contrast, AtCHIL has substitutions of several catalytic residues (Supplementary Fig. 3). Likewise, AtFAP1, AtFAP2 and AtFAP3 encode substitutions at nearly all of the critical catalytic positions of AtCHI, and are devoid of catalytic activity (Supplementary Fig. 3). Studies with GFP fused to full-length FAP1, FAP2 and FAP3 indicate that these proteins localize in the plastid stroma (Supplementary Figs. 4a-d).

We determined X-ray crystal structures of AtCHI, AtCHIL, and the CHI-like domains of AtFAP1 and AtFAP3, and compared these to the previously reported structure of MsCHI8. Despite low sequence conservation, all structures align within 2.3 Åroot-mean-squared deviation for the backbone atoms of equivalent residues (Supplementary Fig. 5). Ligand-binding pockets reside within the helical layer, bounded by two α-helical segments (helix-turn-helix α6-α7 and helical hairpin α4-α5), β-hairpin β3a-β3b, and the face of the core β-sheet, β3a-β3f. Notably, the largest differences in backbone conformations of MsCHI, AtCHI and AtCHIL compared to AtFAP1 and AtFAP3 are confined to the secondary structure elements surrounding the ligand-binding pockets (Supplementary Fig. 5).

The most intriguing finding from the crystallographic structures is that the ligand-binding pockets of the recombinant FAP proteins are occupied by FAs (Fig. 2a-d). Clearly evident in electron-density maps are two abutting molecules of lauric acid (C12:0) in AtFAP1 (Fig. 2a, b), and one molecule of palmitic acid (C16:0) in AtFAP3 (Fig. 2c,d). The ligand-binding site of the FAP proteins encompasses a largely buried, non-polar cavity that sequesters the aliphatic chain(s) of the FAs, and a conserved Arg-Tyr pair that tethers a FA carboxylate group (Fig. 2b, d, and Supplementary Fig. 6). By comparison, CHI and CHIL possess more sterically restricted pockets due to the presence of larger aliphatic residues and an inward shift of the secondary structure elements surrounding their ligand-binding clefts (Figs. 1, 2, and Supplementary Fig. 5). The CHI substrate-binding pocket also includes several polar residues that form key hydrogen-bond interactions with the chalcone substrate (in MsCHI, or with polar nitrate ions in AtCHI) (Fig. 1a, b). The corresponding residues in the FAPs are non-polar. AtCHIL possesses a distinct conservation pattern of the active site pocket (Supplementary Fig. 3). Extraction of ligands with ethanol followed by high performance liquid chromatography–mass spectrometry (HPLC-MS) analyses confirms that all three FAP proteins (including AtFAP2, which was recalcitrant to crystallization) associate with saturated FAs (Fig. 2e). In contrast, with AtCHI and AtCHIL produced under similar conditions, no associated FAs were detected analytically or crystallographically. Exogenous FAs were not supplied during FAP expression in E. coli, purification, or crystallization, so the ligands bound to the FAPs were likely acquired during expression and retained during purification to homogeneity. Indeed, these saturated FAs are among the most abundant FAs in E. coli11.

There are indications that the true ligands of the FAPs in planta may be different FAs. In AtFAP1, the presence of two distinct non-polar tunnels, each occupied by the aliphatic chain of a C12:0 molecule (Fig. 2b and Supplementary Fig. 6a), may indicate a proclivity for longer chain FA recognition. In AtFAP3, the horseshoe-shaped conformation of C16:0 portends a preference for binding a cis-unsaturated C16 or C18 FA, which typically assumes a bent conformation (Fig. 2d and Supplementary Fig. 6b). Notably, AtFAP1 sequesters exogenously-supplied α-linolenic acid (C18:3), which presumably displaces the bound FAs acquired during expression in E. coli (Fig. 2e). We further assessed the binding by the FAPs of a series of common FAs using thermal-stability assays12 (Supplementary Table 1). AtFAP3 interacts with most FAs tested and has maximal relative affinity for C16:0, though C18:0 induces greater thermal stabilization (Supplementary Fig. 7). AtFAP1 preferentially interacts with saturated FAs. However, the strongest stabilizing effect was observed with C18:3 (Supplementary Fig. 8), consistent with the displacement analyses (Fig. 2e) and perhaps a function of AtFAP1 in C18:3 metabolism as indicated also by phenotypic analysis. The thermal stability of AtCHI, included in these analyses as a negative control, was mostly unaffected by the addition of FAs (Supplementary Fig. 9).

We have obtained through a variety of approaches considerable direct and indirect evidence supporting the involvement of AtFAPs in FA metabolism. Network analysis of transcriptomic data13,14 demonstrates maximal expression of AtFAP1 and AtFAP3 in seeds at six days after flowering (Supplementary Fig. 10a), coinciding with the accumulation of storage lipids in the developing embryo15. AtFAP1 and AtFAP3 co-express with genes encoding FA biosynthetic enzymes (Supplementary Fig. 10b). Developmental patterns of AtFAP1, AtFAP2, and AtFAP3 expression were characterized in A. thaliana lines expressing a beta-glucuronidase (GUS) reporter under control of the respective promoters (Supplementary Figs. 11, 12, 13). The FAPs are highly expressed in developing cotyledons, young seedlings, roots, seeds, embryos, macrospores, preanthesis and tapetum. However, while AtFAP2 is expressed throughout the life of the plant, expression of AtFAP1 and especially AtFAP3 is restricted to developing and reproductive tissues.

Homozygous Atfap1 and Atfap2 null plants were propagated from T-DNA insertion lines (Supplementary Figs. 14, 15). These Atfap1 and Atfap2 null plants are indistinguishable from wild-type (WT) plants during vegetative growth (Supplementary Figs. 17, 18), but the Atfap1 mutants show marked differences during reproductive stages. Specifically, Atfap1 siliques are shorter than those of wild type (∼18 mm vs. ∼20 mm) (Fig. 3a), mutant siliques contain 10-20% abnormal ovules (Fig. 3b), and the number of seeds per silique and the yield of viable seeds per plant are reduced in the Atfap1 null lines (Fig. 3c). The AtFAP3-RNAi homozygote lines also present these phenotypic alterations. In addition, the vegetative phenotype of AtFAP3-RNAi is altered, demonstrating early bolting, fast growth, increased branching, reduced apical dominance and reduced overall plant height (Supplementary Fig. 19). No AtFAP3-RNAi line with more than a ∼50% reduction in AtFAP3 RNA (relative to WT) could be identified, nor are any T-DNA tagged Atfap3 knockout lines available. These data thus indicate that a functional AtFAP3 is required for normal plant and embryo development. Double mutants bearing pairwise combinations of homozygous Atfap1, Atfap2, and AtFAP3-RNAi all show additive developmental effects (Supplementary Fig. 18).

Reciprocal crosses between WT and Atfap1 or AtFAP3-RNAi lines point to the maternal inheritance of the aberrant reproductive phenotypes, as an elevated rate of ovule abortion is observed only in crosses in which the maternal parent is a null mutant (Supplementary Figs. 20-21, Supplementary Tables 2-7). The morphological phenotypes of >100 lines of heterozygotic AtFAP3-RNAi were analyzed; these consistently showed a restoration of the WT vegetative phenotype (Supplementary Fig. 22). However, the aberrant reproductive phenotype of the AtFAP3-RNAi mutant was maintained, indicating that phenotypic effects observed in the AtFAP3-RNAi lines are due to the specific knockdown of this gene. Analysis of FA composition reveals that Atfap1 null lines have elevated total fatty-acid levels in both leaves and seeds relative to WT (Fig. 3d, e and g). These phenotypic alterations in leaves are temperature dependent (apparent only in plants grown at 15°C and 22°C) and due primarily to an increased level of the lower-melting-temperature unsaturated FA, C18:3. The FA content and distribution in leaves are indistinguishable between WT and mutant plants grown at 26°C (Fig. 3f). Similarly, AtFAP3-RNAi plants and Atfap double mutants have altered FA composition and increased FA content in leaves and seeds (Supplementary Fig. 23). This temperature-dependent phenotype reflects a decrease in the more fluid acyl chain component of trienoic FAs that accompanies increasing temperature16,17.

We identified three relatives of CHI in Arabidopsis which appear to be FA-binding proteins of undetermined function. Phylogeny and sequence analyses indicate that the CHI fold occurs in most eukaryotic lineages (with the notable exception of animals) and even in bacteria. CHIL, as a divergent relative of FAP3, initially arose in mosses and may have served as the ancestor of enzymatic CHI in vascular plants (Supplementary Figs. 2 and 3). This ancestry for plant CHI clarifies the mystery surrounding the identity of the progenitor protein, and serves as a model to understand adaptation of a ligand-binding protein to a highly efficient and stereospecific enzyme. An evolutionary connection between fatty acid and flavonoid metabolism18 is also evident from the structural and mechanistic similarities between chalcone synthase (a type-III polyketide synthase (PKS)) acting one step upstream of CHI in the flavonoid pathway and ketoacyl-synthase III (KAS III) involved in chloroplast and bacterial fatty-acid biosynthesis. Together, CHI and CHS provide the foundation to examine the evolution of more recent metabolic pathways and metabolite classes from widespread biosynthetic pathways of primary metabolism.


Methods Summary
Structural biology

Crystal structures of the CHI domains of AtCHI, AtCHIL, AtFAP1, and AtFAP3 were determined as described in Supplementary Materials and Methods and summarized in Supplementary Table 8.

In vitro biochemistry

CHI activity was assayed5,7. Ligands bound to the purified AtFAP proteins were extracted with ethanol and analyzed by reversed-phase HPLC-MS. The effects of fatty acids (FAs) on the melting temperature of the AtFAP proteins were measured using a Thermofluor-based assay12.

Phylogenetic and sequence analyses

Homologs of AtCHI were identified by iterative psi-blast and profile Hidden Markov Model (HMM). Alignments were curated using protein structure superpositions and homologous groups determined manually.

Informatics

Co-expression patterns of A. thaliana genes encoding FAP proteins and genes associated with FA biosynthesis were evaluated with data compiled from 72 microarray experiments using MetOmGraph (www.metnetdb.org)13.

Plant growth and mutant analyses

Plants were grown in random block designs at 22°C under 16 hr light/8 hr dark. FAs were measured 5 hr after onset of light. Temperature effects were observed after 10-d growth at the specified temperatures. Homozygous Atfap1-1, Atfap1-3, Atfap2-1 and Atfap2-2 mutant alleles (Supplementary Fig 14 and Supplementary Table 3) were generated from SALK_130560, SALK_039829, SAIL_171_C12 and SAIL_616_D09 (Arabidopsis Biological Resource Center, ABRC) stocks, respectively. Atfap3 RNAi lines (Supplementary Fig. 16 and Supplementary Table 9) were generated from A. thaliana Col-0 plants, transformed with Agrobacterium tumefasciens strain GV3101 containing vector CATMA1a44560 (Nottingham Arabidopsis Stock Center, NASC)19. AtFAP RNA was quantified by RT-PCR (Supplementary Figs. 15 and 16). Transgenic A. thaliana lines were generated with AtFAP1, 2, 3 promoters or promoter + cTP /or + CDS fused to GUS and green fluorescent protein (GFP).

FA quantification in plants

FAs were extracted from plant tissues using barium hydroxide hydrolysis20, modified by methylation and analyzed by gas chromatography–mass spectrometry (GC-MS). Nonadecanoic acid was used as internal standard and a reference FA mixture was used for calibration and retention time determination (plantmetabolomics.org).



Notes

FN3Supplementary Information is linked to the online version at www.nature.com/nature.

FN4Author Contributions M.N.N. R.N.P., and G.V.L. made equivalent contributions and should be considered joint first authors. M.N.N. experimentally characterized the FAP genes in planta. M.E.B., R.N.P. and E.L. expressed, purified and crystallized proteins. L.L. designed genetics experiments and constructs. G.V.L. and F.P. carried out fatty acid binding analyses and solved the x-ray crystal structures. R.N.P. carried out thermal-shift assays of fatty acid binding. G.M. and E.L. performed phylogenetic and sequence analyses. J.P.N. designed the biochemical experiments; E.S.W. designed the bioinformatics and functional genomics experiments. The manuscript was written by R.N.P., G.V.L., M.N.N., J.P.N., G.M. and E.S.W.

FN5Author Information Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 4DOI (AtCHI), 4DOK (AtCHIL), 4DOL (AtFAP3), and 4DOO (AtFAP1).

FN6Reprints and permissions information is available at www.nature.com/reprints.

FN7The authors declare no competing financial interests.

We thank A. Perera, B. Nikolau, H. Ilarsan and J. Peng for technical training (to M.N.N.), J. Peng for the fap1-1 homozygote mutant line, D. Nettleton & H. Wang for statistical analysis of seed FA data, and Eric Scheeff for assistance in Bayesian phylogenetic analysis. This research was supported in part by a Fulbright Fellowship (M.N.N.). This material is based in part upon work supported by the National Science Foundation under Award No. MCB-0645794 (J.P.N.), EEC-0813570 (E.S.W.), MCB-0951170 (E.S.W.), and by the National Cancer Institute Award No. CA14195 (G.M.). J.P.N. is an investigator with the Howard Hughes Medical Institute. Portions of this research were conducted at the Advanced Light Source (ALS) a national user facility operated by Lawrence Berkeley National Laboratory, on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The Berkeley Center for Structural Biology is supported in part by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences. We thank the staff at the ALS for assistance with X-ray data collection. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.


References
1. Hartmann T. From waste products to ecochemicals: fifty years research of plant secondary metabolismPhytochem6828312846Year: 2007
2. Ferrer JL,Austin MB,Stewart C Jr,Noel JP. Structure and function of enzymes involved in the biosynthesis of phenylpropanoidsPlant Physiol Biochem46356370Year: 200818272377
3. Peer WA,Murphy AS. Flavonoids and auxin transport : modulators or regulators ?Trends Plant Sci21556563Year: 200718198522
4. Bednar RA,Hadcock JR. Purification and Characterization of Chalcone Isomerase from SoybeansJ Biol Chem26395829588Year: 19883384815
5. Jez JM,Noel JP. Reaction mechanism of chalcone isomerase: pH dependence, diffusion control, and product binding differencesJ Biol Chem27713611369Year: 200211698411
6. Albery WJ,Knowles JR. Evolution of Enzyme Function and the Development of Catalytic EfficiencyBiochem1556315640Year: 1976999839
7. Jez JM,Bowman ME,Noel JP. Role of hydrogen bonds in the reaction mechanism of chalcone isomeraseBiochem4151685176Year: 200211955065
8. Jez JM,Bowman ME,Dixon RA,Noel JP. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomeraseNat Struct Biol7786791Year: 200010966651
9. Gensheimer M,Mushegian A. Chalcone isomerase family and fold: no longer unique to plantsProtein Sci13540544Year: 200414718655
10. Shirley BW,Hanley S,Goodman HM. Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutationsPlant Cell4333347Year: 19921354004
11. Oursel D,et al. Identification and relative quantification of fatty acids in Escherichia coli membranes by gas chromatography/mass spectrometryRapid Commun Mass Spectrom2132293233Year: 200717828792
12. Niesen FH,Berglund H,Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stabilityNat Protocols922122221Year: 2007
13. Mentzen WI,Wurtele ES. Regulon Organization of ArabidopsisBMC Plant Biol899Year: 200818826618
14. Mentzen WI,Peng J,Ransom N,Nikolau BJ,Wurtele ES. Articulation of three core metabolic processes in Arabidopsis: fatty acid biosynthesis, leucine catabolism and starch metabolismBMC Plant Biol87690Year: 200818616834
15. Ruuska SA,Girke T,Benning C,Ohlrogge JB. Contrapuntal networks of gene expression during Arabidopsis seed fillingPlant Cell1411911206Year: 200212084821
16. Falcone DL,Ogas JP,Somerville CR. Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid compositionBMC Plant Biol41731Year: 200415377388
17. Murakami Y,Tsuyama M,Kobayashi Y,Kodama H,Iba K. Trienoic fatty acids and plant tolerance of high temperatureScience287476479Year: 200010642547
18. Austin MB,Noel JP. The chalcone synthase superfamily of type III polyketide synthasesNat Prod Rep2079110Year: 200312636085
19. Bonaventure G,Salas JJ,Pollard MR,Ohlrogge JB. Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growthPlant Cell1510201033Year: 200312671095
20. Hilson P,et al. Versatile gene-specific tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applicationsGen Res1421762189Year: 2004
21. DeLano WL. The PyMOL Molecular Graphics SystemDeLano ScientificSan Carlos, CA, USA http://www.pymol.org. Year: 2002
22. Wallace AC,Laskowski RA,Thornton JM. LIGPLOT: A program to generate schematic diagrams of protein-ligand interactionsProt Eng8127134Year: 1995
23. Collaborative Computational Project Number 4The CCP4 suite: programs for protein crystallographyActa CrystallogD50760763Year: 1994

Article Categories:
  • Article


Previous Document:  The complex of tmRNA-SmpB and EF-G on translocating ribosomes.
Next Document:  Asymmetric hydrogenation of imines with a recyclable chiral frustrated Lewis pair catalyst.