Kinemages: make your own molecules for teaching

9,

29Dedjwd,B.etBJ.(19941 C&/76, 1025-1037 30 Sun, H.. Ctmdes.

C. Ii., Lau, L. F. and

31 32

E., Zhelev, N., l-lamiii, C. A. and ) L. c. (1994) w. c&I 6w. 14, i06&1074

33 m.

R.A.,PiQpUd,V.and

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44 Sanchez, I. era!. (1994) 794-798

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45 Yan,M.etal.(1994)iU&m372,

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#otdhgim,k.(1993)EkcBol. 12.2377.2387

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. A., Zhekv, N. and

34 1. and Tiehan,

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36 Ghci, K. Y., SattWrg, B., Ly#ns, 0. M. md f?H78,499-512 Elan, E. A. (1 37 Hsiao, K. M., * S. Y., Shih, S. 1. and Ferrell, J. E. (1994) fkc. h&t1 kad. Sci. USA 91, 5480-5484 38 Mwsuda,S..Gotoh, Y.artd Nbhida, E. (1993) 1. Bids Cbem.268.3277-3281 39 Dewy, Y.,Gottlieb, R. A., Smaal,T. and (l~)~a~~7~,~06~-~~91 40 -Viiiana,P. etaf. (1994) Nature 370, 527-532 41 &guski.M.S.andfMhrmick,F.(1993) #arufe 366,643-654 42 Wu, 1. et sl. (1993) Science 262, 1065-1069 43 Treisman, R. (1994) Cvrr. Opin. Genet. Dev. 4,

35 Davis, R. J.

Derij8rck 8.. Wu, 1. Ii. and 265,8%-8D8

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46 Mindan, A. eta/. (1994)ScMce266, 1719-1723

(1994) Meme

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Kinemages: makeyourown molecules forteaching

text. The synthesis and utilization of Mnemages by students is also easily incorporated into introductory biology

andbiochemistry courses. Kinemages are also usefulas ready

references in laboratory research. The three-dimensional structure of a protein under investigation can be viewed the softwareprograms.For example, using MAGE without the aid of an exFig.1 showsthe u-carbonbackbone of pensive molecular modeling workstation. spinach ferredoxfn reductasewith The Mnemages created during the boundflavinadeninedinucieotideand course of research or as the result of an 2’-phospho4MMP? Thisffgurewasgen- origin4 structural characterization study eratedin than five minutes using can then be used to illustrate the resultthe built-i tipts option of PREKfN ing milnuscript in Pro&& Scitmce. and the coordinates file of ferredoxin Accompanying each issue of this jourreductase, whichwastransferred from nal is a diskette containing kinemages the Brookhaven PDB(Internetprotocol that are associated with the publfshed address130.199.144.1). No addltfonaf papers*

editing of the figure was requfred, aktmgh many optkms to customfze lchmges are available(seefolIowing The first step in generating a exampfe). Thestructurecanbe rotated kfnemage is to accessthe PDB fflethat by pofntfngand draggtngwfth the contains the coordinates of the u the screencan be split for mokule. For Ffgs 1 and 2 this was stereoview&g and subunitscan be accompifshed by usingffletransferproturnedon/off with the button panel tocol (ftp). Oncethe ffles havebeen (rightsideof Pfgs1and2).Additfonaffy,locatedon the PDB server they can be fndfvfdual atom labels, dfstances transferredto your computervfa the betweenatomsanddihedralanf#escan ‘get’ command.fhe exact commands be displayedby simply pointfngand for locatingand transferringffles are ckkhgtith the mouse. eo?nputer-system specific,so you may Thfssoftware,wfth its inherentease needhelp from your local computer systemmanager. OncetherelevantPDf3fffeis on your desktopcomputer, theinitialgeneratfon of a kinenrage is as simpleas opening the PfEKlNprogram,sefectingthe fife -WtURandhmction.Howerer,~ to be translatedinto a kfnemage, and UsemwandMAGE fohowfng athreestepprocessprompted dguresfor the materfaf bytheprogram.ff youareusingPREKfN coversdfMnyCourse,regardfessofthe for the first time, selectingthe first 0 1995, EMvler

Science Ltd 0968~0004/95/$09.50

MAGE and PREKlN also allow for the constructfon of more complex ilhrstrations, Such figures may include Is, presd views, and ‘animation’ between one or more conformers of a structure. Figure 2 shows a more detailed kinemage, the Cro repressor protein bound to DNA4. However, as informative as this kieneration was still straightforward. A kinemage file can be edited using a word processing program. Although the file, with its many lines of numbers, may look confusing, only the command lines that begin with the ‘@, symbol are actually edited. These lines designate the The kwaben bztckbm of spine& fe bound ligands button labels, colors, views, etc. of the ed; fiiwine he entire corn(2”-PfJ ~IX?IXI bai puter &de scales to structures displayed by MACE. The the right. The kinemage was produced from the fW file 2MR using the PREKiN program. blocks of numbers below the command The IMAGEscreen was then captured and PriMed w& a colorlaserprinter. lines describe the vectors associated with that piece of the kinemage. These blocks, with their command lines encountered. Although the MACE proattached, can be cut or moved to For the illustration of biomolecules gram has some limitations, such as delete or alter the structures of the on a personal computer, PRFKIN and ‘jumpy’ rotation of structures that MAGE are the most versatiie yet include a large number of vectors kinemage. In older versions of MACE and easy to master programs that I have (>400), the tremendous advantages this PKEKIN,in order to change the button hierarchies. structure color, atom labels, etc. it was necessary to identify the individual command line in the Wnemage file and edit it using a word processor. However, recent updates to the MACE program allow for many of these kinemage features to be edited on screen. Vectors can be added, deleted or moved, atom labels and structure colors can be changed, and preset viewing angles can be created: all from the ‘Edit’ pulldown menu in the MAGE program. For example, the view shown of the Cro repressor protein (Ffg. 2) was created and saved simply by opening the Mnemage file in the MAGE program, rotating the structure to its desired position, and choosing the ‘Update View’ command from the Edit menu. The color of the DNA sugarphosphate backbone was changed from blue to green by using the ‘pickshow’ option in the Edit menu. With the fncluslon of the on-screen editing of 2 kinemages through the MAC8 program, The Cro mpressot protein bound to DNA(PO6fite XRO). The colors, view and @MS were what was once a refatlvely easy editing option in MAGE. Cotor coding is editedusingawofdfmmssorandtlwonscreen editing process has become exquisitely as fobws: Cro pro&in dimer, red; DNA sugar-phosph~ bzHbon@. green; A-T base Pairs, pale blue; G-C base pairs, deep blue. simple.

l23

publication. electronic Pr&in-2-5.txt, and CookJin.btt describe in varying detail the use of the two programs. Demo3Jakin and Demo3Jbkin are files that contain excellent demon&rations of what can be created with ~~~by~~~~r, the most important files available from the elecmnk lw&l Scienceare those that contain the programs themselves. It is not often that such a powerful

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the use of this software to illustrate

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made available to the entire b&hem istry conmumity free of charge.

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2 Richardson, 0.6. and Richardson, Sci. 1,3-9 2 Richardson, 0. C. and Richardson,

Protein

J. S. (19921 J. S.

(1994)TrendsBiochem. sci. 19, 135-138 3 Kafplus, P. A., Daniets, M. 1. and Herriott,

1. R.

(1991) Scieme251,EW

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Department of Chemistry and Biochemistry, Den&on University, Granville, OH 43023,

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email: sokoMkc.denison.edu

although different concentrations in the secondary PCR also gave a different type of smear In addition, changing the annealing temperature, the amount of poiymerase or the number of cycles in either the primary or the secondary Reamplfficationof PCRfragments PCRdid not correct the problem. column that highlights currant discussions in Unfortunately, this poor netter susavahble on the Internet. This month’s column pected that the problem was a combiwhile trying to reamplify DNA made by nation of all the various parameters dispertake in the nawsgroup, see the accussed, and he continued to experience box the smears. Someone su Andrew lbiymke (zool2Ol&sc.canter- template for reamplification of the try ‘touchdown’ PCR, a method by desired DNA fragment - smears were which the annealing temperature is .nz)wastryingtoampllfya1kb ofmltochondrialDNAfroma seen on an agarose gel every time. In incrementally lowered during each human sperm cell using primers ‘Frohmanlan’ terms, this common result cycle of the PCR, and that this might speclflc to the target gene. Since the is often referred to as ‘ampii-schmutz’i help to determine the anneaiing temstartiqj template was of such low quart- and is mostly caused by nonspecific perature at which a more specific pritlty and the band of interest was nearly amplification in the original PCR. mary product can be generated3”. erthe und of the It is also likely that the smears are Someone else suggested that chop chain by using far too much DNA in ping up the smear with restriction (?pcQ* he ~~~~~~ PCR Reampllfication enzytxs known not to cleave within the iLz?m to inem the yield of the works well when the concentration of PCR product of Merest might help altemplate DNA ls quite low. Therefore, leviate the smears. Another way would difndm. the primary PCR product must be di- be to design nested primers, which are luted at least 10 OOO=fold before reampli- complementary to sequences that lie fkath. However, even when the prl- within the fragment generated by the mazy mixture of his DNA was diluted first round of amplifications. down to as We as 0.1 pg of template If a DNA band of the expected size DNA&3mearwasnotresolvedlntoa can be seen on a gel as well as a backdiscreteband. ground of smeared DNA, an effective Short of suggesting that he should method used to improve reamplifidesign a new set of ollgonucleotide cation yield and eliminate most of the primers more specific for the target unwanted products is gel purification ar with the problem or size fractionation of the band of might improve the interest. Since slicing a small piece of polythe inkiai ~~~ am&km. For acrylamide gel is very difficult to do, -pkthtsmrvybedonebyLnneasone person suggested that he extract ter+xature, or by the DNA from the gel by cutting a * concentration, the trough just ahead of the m&rating DNA number of cycles, the amount of en- band and filling it with low-meltingzyme, or the concentration of dNTPs2. point agarose. Electrophoreslng the gel WMly the h&f+ concentration was horizontally at approximately 10 V cm-* opthkd for the primary PCR, for MOmin in a submarine-style

Methods andreagents