Gli autori desiderano ringraziare il personale PXRR al NSLS (Brookhaven National Laboratory) per l&#39;assistenza durante la raccolta di dati e Monica Pillon aiuto per la purificazione del DNA. Questo lavoro è stato sostenuto dal Canadian Institutes of Health Research (MOP 67189).

1. Protein Purification Il linker flessibile che collega la N-(oligomerizzazione) e C-terminale (DNA binding) dominii Seqa aiuti riconoscimento dei hemimethylated ripetizioni GATC separate da 1-3 giri sul DNA. Per questo studio, abbiamo utilizzato una variante dimerica della Seqa (SeqAΔ (41-59)-A25R) con una mutazione puntiforme nel dominio N-terminale che impedisce l&#39;ulteriore oligomerizzazione e un linker accorciata che limita legante il DNA a tandem GATC ripete separati esclusivamente un giro sul DNA (Figura 1) 2,8. <ol><li> Trasformare BL21 (DE3) con il plasmide codificante Seqa sotto il controllo del promotore T7, </li><li> Piastra la reazione di trasformazione in piastre di agar LB-tra 100 pg / ml ampicillina, </li><li> Selezionare colonie miste per inoculare una piccola coltura overnight (media LB con 100 pg / ml ampicillina), </li><li> La mattina dopo inoculare un 1 l di media utilizzando una diluizione 1:100 del overniGHT cultura, </li><li> Crescere le cellule ad una OD 600 di ~ 0,7 e indurre la produzione di proteine ​​per aggiunta di isopropil β-D-1-tiogalattopiranoside (IPTG) ad una concentrazione finale di 1 mM, </li><li> Continuare l&#39;incubazione per 3 ore a 37 ° C con agitazione orbitale e quindi raccogliere le cellule per centrifugazione (10 &#39;a 3300 g), </li><li> Risospendere il pellet di cellule in tampone A e purificazione lisi mediante sonicazione, </li><li> Azzerare il lisato tramite centrifugazione (40 min a 39.000 g) e caricare il surnatante su una colonna di eparina equilibrata con tampone di purificazione, </li><li> Eluiscono Seqa usando un gradiente lineare di NaCl 1 M (Seqa eluisce a ~ 0,7 M NaCl), </li><li> Pool Seqa le frazioni contenenti insieme, diluire per abbassare la forza ionica del campione e di carico in una colonna a scambio cationico cromatografia equilibrata con tampone di purificazione, </li><li> Usando un gradiente lineare di sale, puro Seqa eluisce a ~ 0,4 M NaCl, </li><li> Pool i Seqa contenenti frazioni insieme, concentrate (3 mg / ml) e memorizzare in tampone di conservazione. </li></ol> 2. Purificazione del DNA <ol><li> Ordine oligonucleotidi complementari non metilato e metilati dalla vostra azienda preferita, </li><li> Sciogliere 1 pmol di ogni singolo filamento di DNA liofilizzato in 800 ml di autoclavato DDH 2 O, vortice e lasciate riposare per 10-20 min, </li><li> Aggiungere 800 pl di tampone di caricamento 2X preriscaldato a ciascun oligonucleotide, </li><li> Oligonucleotidi per 20-30 nucleotidi, preparare una grande gel 10% denaturante (160 x 250 x 1 mm): * Mescolare bene 80 ml di miscela PAGINA 10%, 80 TEMED ul e 800 ul persolfato di ammonio per gel e versare, * Una volta polimerizzato, rimuovere il pettine e lavare i pozzetti con DDH 2 O a fondo, * Montare i gel su gel colato tra piastra di raffreddamento e riempire i serbatoi superiore e inferiore con tampone di corsa (1X TBE), * Pre-eseguire il gel a 700-750 V per riscaldare il gel fino a 55 ° C, * Fermare la corsa e sciacquare il Thoro pozziughly con tampone di corsa. </li><li> Riscaldare gli oligonucleotidi a 90 ° C per 2 min, </li><li> Vortex e centrifugare i campioni e subito prima di caricare il gel, </li><li> Attivare il gel a ~ 700 V e fermarlo una volta che il oligonucleotide è migrata a metà strada. (Si noti che su un gel di poliacrilammide 10% blu di bromofenolo co-migra con oligonucleotidi ~ 20 basi di lunghezza e xilene cyanol FF con ~ 60 basi di lunghezza), </li><li> Arrestare il gel, smontarlo dal box gel e rimuovere i distanziatori, </li><li> Su una superficie piana, rimuovere una piastra di vetro e coprire il gel con pellicola trasparente, </li><li> Girare il gel intorno, rimuovere l&#39;altra piastra di vetro e coprire con pellicola trasparente, </li><li> Contrassegnare le bande utilizzano la luce UV e una piastra fluorescente dietro il gel per vedere l&#39;ombra del DNA, </li><li> Tagliare la band con una lama di rasoio in piccoli pezzi e trasferirli in una provetta sterile 15 ml, </li><li> Aggiungere 9 ml di tampone di eluizione ed eluire la notte a 37 ° C con agitazione, </li><li> Attenzione trasferire la soluzionead un tubo da centrifuga sterilizzato utilizzando un gel-loading punta della pipetta per evitare pezzi acrilammide trasferimento e aggiungere 1 ml di acetato di sodio 3 M a pH 7 (diluizione 1:10) più 25 ml di etanolo al 100% refrigerati (2,5 volumi), </li><li> Incubare a -20 ° C per almeno 3 ore, </li><li> Centrifugare e trasferire il surnatante in un tubo separato, </li><li> Asciugare il pellet sulla velocità-vac a fuoco medio, </li><li> Risospendere il pellet in 400 ml di autoclavato DDH 2 O e il trasferimento in una nuova provetta, </li><li> Aggiungere 40 ml di acetato di sodio 3 M a pH 7 e 1 ml di etanolo al 100%, mescolare bene (vortex) e incubare per 30 min a temperatura ambiente, seguito da 30 minuti a -20 ° C, </li><li> Centrifugare per 15 minuti a 18.000 g ed eliminare il surnatante, </li><li> Risciacquare il precipitato con 100 ml di etanolo al 70% freddo per rimuovere residui di sale dal pellet e centrifuga per 6 minuti a 18.000 g. Eliminare l&#39;etanolo e asciugare il pellet sulla velocità-vac, </li><li> Risospendere il pellet in un totale di 100 pl di autoclavato DDH <sub&gt; 2 O. Misurare la concentrazione del oligonucleotide, </li><li> Per temprare i duplex di DNA hemimethylated, mescolare concentrazioni equimolari dei singoli filamenti complementari e le miscele riscaldare a 95 ° C in un bagno d&#39;acqua per 5 minuti e poi lasciar raffreddare lentamente a temperatura ambiente all&#39;interno del bagno d&#39;acqua. </li></ol> 3. Proteina-DNA Complex Formazione e analisi <ol><li> Miscelare volumi uguali di acqua di SeqAΔ (41-59)-A25 (81 mM) e DNA hemimethylated (81 mM), </li><li> Incubare a temperatura ambiente per 15 minuti e conservare a 4 ° C fino al momento di usarlo, </li><li> Schermo per condizioni di cristallizzazione che utilizzano commerciali sparse schermi a matrice, </li><li> Una volta conduce cristallizzazione iniziali sono stati identificati, di ottimizzare le condizioni di crescere cristalli qualità diffrazione, </li><li> Cryoprotect le risultanti Seqa-DNA cristalli, o aumentando la quantità di PEG 400 presenti nella soluzione di cristallizzazione ad una concentrazione finale del 25% (v / v) o aggiunta di glicerolo al 20% (v / v) alla soluzione di cristallizzazione, </li><li> Scoop singoli cristalli con un anello di nylon, e li flash-congelamento in azoto liquido, </li><li> Verificare il limite di diffrazione di ogni cristallo a 100 K. </li></ol> 4. Risultati rappresentativi Per ottenere la struttura cristallina di SeqAΔ (41-59)-A25R legato al DNA hemimethylated, abbiamo ottimizzato consecutivamente tre parametri della DNA: (i) separazione tra hemimethylated sequenze GATC; (ii) lunghezza del duplex, e (iii) l&#39; assenza / presenza di 5 &#39;sporgenze. Electro-mobilità saggi spostamento indicano che SeqAΔ (41-59)-A25R si lega preferenzialmente ripete GATC separati da 9-10 paia di basi (Figura 1). Pertanto, inizialmente proiettato su due piani 23-24 paia di basi (pb) lunga che contiene due sequenze hemimethylated GATC separati da 9 o 10 bps. Tre appartamenti su due piani prodotto cristalli ben sagomate (Figura 2). Alse nessuno dei cristalli diffratta di alta risoluzione, il 23 bps duplex lungo con i due siti GATC separati da 9 bps diffratta raggi X migliore del resto, che indica che una separazione GATC di 9 bps è preferito per la cristallizzazione. Pertanto, abbiamo fissato l&#39;inter-GATC spaziatura a 9 bps per tutte le schermate successive. Generalmente, cristallizzazione DNA è favorito per lunghezze duplex corrispondenti esatte spire elicoidali perché molecole di DNA più possibile impilare testa-coda per formare un continuo B-DNA all&#39;interno del cristallo 9. Pertanto, abbiamo ridotto la lunghezza complessiva dei duplex a 21 bps (cioè due spire elicoidali). Mentre un duplex 21 bps con estremità smussata non ottenere cristalli qualità diffrazione (dati non mostrati), un duplex 21 bps con una sola sporgenza 5 &#39;nucleotide su ciascuna estremità produssero cristalli che diffratta a 5 Å nella nostra sorgente casa. Il miglioramento del limite di diffrazione suggerito che end-to-end duplex associazione è stata effettivamente favorireing cristallo imballaggio. Poiché Seqa interagisce con sequenze GATC che sono sulla stessa faccia del DNA, la faccia opposta del duplex DNA deve essere esposta al solvente. Per migliorare ulteriormente i cristalli del complesso, abbiamo poi modificato la sequenza del duplex per includere un dinucleotide CG tra i due siti GATC promuovere scanalatura-backbone interazioni con molecole di DNA adiacenti nel cristallo attraverso la faccia opposta del duplex, un metodo che è stato usato per migliorare cristallizzazione DNA in passato 10. Tuttavia, il limite di diffrazione dei cristalli cresciuti con la CG-duplex contenente era identico a quelli coltivati ​​con un duplex DNA simile che non conteneva il dinucleotide CG (Figura 3). Questo risultato indica che scanalatura-dorsale interazioni non sono importanti in questo caso. Successivamente abbiamo ottimizzato la lunghezza delle sporgenze confrontando i cristalli cresciuti con un duplex DNA che aveva due nucleotidi addizionali ad ogni5 &#39;. Questo cambiamento ha avuto un effetto drastico sulla morfologia cristallina, così come, limite di diffrazione che indica che il nucleotide complementare drammaticamente cambiato i contatti molecolari e organizzazione cristallo maggiore (Figura 3). La struttura cristallina di SeqAΔ (41-59)-A25R legato a questo duplex DNA scorsa ha confermato che la faccia libera del DNA duplex non è attiva nei contatti di cristallo, come previsto dal limitato effetto di introdurre una CG-dinucleotide. Nonostante la dimensione relativa di proteine ​​e DNA, la maggior parte delle interazioni tra compagni di simmetria sono mediati dalla proteina-proteina e proteina-DNA interazioni (Figura 4). È interessante notare, in questo caso particolare, l&#39;effetto benefico di sbalzo un dinucleotide 5 &#39;non è dovuto alla formazione di una pseudo-continuo DNA. Invece, l&#39;estremità 5 &#39;del filamento progetti metilate distanza dagli assi DNA e interagisce con la molecola Seqa prossimale del complesso, spiegando perché due nucleotidi w<html> ere strettamente necessario per modificare il cristallo di imballaggio 8,11. <img alt="Figura 1" src="/files/ftp_upload/4266/4266fig1.jpg" /> Figura 1. Il legame di SeqAΔ (41-59)-A25R al DNA hemimethylated. (A) Rappresentazione schematica dei domini di Seqa che oligomerizzazione di proteine ​​mediato e legame al DNA. (B) elettroforetica spostamento saggio mobilità dei SeqAΔ (41-59)-A25R con DNA che contengono due sequenze hemimethylated GATC separati da un numero crescente di coppie di basi ( X). La corsia più a sinistra (etichettato Bianche) contiene una miscela equimolare dei DNA con 5, 7, 12, 21, 25 e 34 coppie di basi tra le due sequenze GATC in assenza di SeqAΔ (41-59)-A25R. (C) diagramma illustrante le tre variabili ottimizzati sulle duplex di DNA per ottenere cristalli di qualità diffrazione. upload/4266/4266fig2.jpg &quot;fo: content-width =&quot; 6in &quot;fo: src =&quot; / files/ftp_upload/4266/4266fig2highres.jpg &quot;/&gt; Figura 2. Effetto di variare la distanza tra GATC. Sintesi degli oligonucleotidi utilizzati e cristalli ottenuti con 23-24 appartamenti su due piani bp lunghi contenenti due siti hemimethylated GATC separati da 9 o 10 paia di basi. Tutte le immagini dei cristalli sono state scattate allo stesso ingrandimento e la barra della scala indica 100 micron. Il limite di risoluzione di ogni SeqAΔ (41-59)-A25R-DNA cristallo si basa su immagini di diffrazione raccolti su di un RU-300 Rigaku raggi X sistema generatore. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 3" src="/files/ftp_upload/4266/4266fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4266/4266fig3highres.jpg" /> Figura 3. Effetto della variazione della DNA termina. Sintesi di tegli oligonucleotidi utilizzati e cristalli ottenuti con 21 bps lunghi duplex contenente due siti hemimethylated GATC separati da 9 coppie di basi e di cui 0, 1 o 2 nucleotidi 5 &#39;sporgenze finali. Tutte le immagini dei cristalli sono state scattate allo stesso ingrandimento e la barra della scala indica 100 micron. Il limite di risoluzione di ogni SeqAΔ (41-59)-A25R-DNA cristallo si basa su immagini di diffrazione raccolti a linee di luce e X12C X29 (NSLS, BNL). <a href="http://www.jove.com/files/ftp_upload/4266/4266fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 4" src="/files/ftp_upload/4266/4266fig4.jpg" /> Figura 4. Imballaggio di cristallo di SeqAΔ (41-59)-A25R legato al DNA con sbalzo dinucleotide: Visto di (A) in alto e il lato (B). L&#39;unità asimmetrica contiene due SeqAΔ (41-59)-A25R-DNA in cui viene mostrato la proteina in grigio mentre il DNA è indicato in arancione. Simmetricory correlato SeqAΔ (41-59)-A25R-DNA molecole sono presenti in bianco per la proteina e giallo per il DNA. Questo dato è stato preparato utilizzando PyMOL 12. Questo dato è legato al filmato 1. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig4large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . Movie 1. <a href="http://www.jove.com/files/ftp_upload/4266/4266movie1.avi" target="_blank">Clicca qui per visualizzare filmati</a> .

<ol>
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Nessun conflitto di interessi dichiarati.

Una delle maggiori sfide nel macromolecolare cristallografia a raggi X è l&#39;ottenimento di cristalli di qualità di diffrazione. Nel caso di complessi proteici o proteine-DNA, questa sfida è aggravato a causa delle variabili aggiuntive che devono essere ottimizzati. Si crede che la lunghezza del DNA e la presenza di sbalzi appiccicose per migliorare associazione di molecole di DNA più vicini in un pseudo-duplex sono i principali parametri per ottimizzare. Tuttavia, abbiamo dimostrato che la natura e la lunghezza d...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Catalogo # </td><td> Commenti (opzionale) </td></tr><tr><td> TRIS </td><td> Bioshop </td><td> TRS003.5 </td><td></td></tr><tr><td> Acido etilendiamminotetraacetico (EDTA) </td><td> Fisher Scientific </td><td> E478-500 </td><td></td></tr><tr><td> Dithiotheitrol (DTT) </td><td> Bio Base Inc. </td><td> DB0058 </td><td></td></tr><tr><td> NaCl </td><td> Bioshop </td><td> SOD002.10 </td><td></td></tr><tr><td> Glicerina </td><td> Caledon </td><td> 5350-1 </td><td></td></tr><tr><td> Saccarosio </td><td> Sigma-Aldrich </td><td> S5016-500G </td><td></td></tr><tr><td> Di sodio dodecil solfato (SDS) </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td> Urea </td><td> Bioshop </td><td> URE001.5 </td><td></td></tr><tr><td> 40% Bis 29:1 / acrilammide </td><td> Bio Base Inc. </td><td> A0007-500ml </td><td> Conservare a 4 ° C </td></tr><tr><td> Acido borico </td><td> EMD </td><td> BX0865-1 </td><td></td></tr><tr><td> Xilene cyanol FF </td><td> Bio-Rad </td><td> 161-0423 </td><td></td></tr><tr><td> Blu bromofenolo </td><td> Bioshop </td><td> BR0222 </td><td></td></tr><tr><td> Doppio sistema di regolazione verticale Gel </td><td> CBC Scientific Company Inc. </td><td> DASG-250 </td><td></td></tr><tr><td> Indice cristallizzazione schermo </td><td> Hampton ricerca </td><td> HR2-144 </td><td> Conservare a 4 ° C </td></tr><tr><td> I cristallizzazione guidata schermo </td><td> Emerald BioSystems </td><td> EBS-WIZ-1 </td><td> Conservare a 4 ° C </td></tr><tr><td> Wizard II cristallizzazione schermo </td><td&gt; Emerald BioSystems </td><td> EBS-WIZ-2 </td><td> Conservare a 4 ° C </td></tr><tr><td> Classics cristallizzazione schermo </td><td> Qiagen </td><td> 130701 </td><td> Conservare a 4 ° C </td></tr><tr><td> Intelliplate vassoi </td><td> Art Robbins Instruments </td><td> 102-0001-00 </td><td></td></tr><tr><td></td><td></td><td></td><td> Soluzioni Proteine ​​tampone purificazione: 100 mM TRIS pH 8, 2 mM EDTA, 2 mM DTT e 5% glicerolo. Proteine ​​tampone di conservazione: 20 mM TRIS pH 8, 150 mM NaCl, 5 mM DTT, 0,5 mM EDTA e il 5% glicerolo. Gel mix caricamento: Aggiungere 20 g di saccarosio, 25 mg di blu di bromofenolo, 25 mg di xilene cyanol FF, 1 ml di 10% w / v SDS e 10 ml di TBE 10X a 70 ml di autoclavato DDH 2 O. Mescolare con blando riscaldamento fino al saccarosio è sciolto e regolare la finaleil volume a 100 ml con autoclavato DDH 2 O. Conservare a 4 ° C. Loading buffer 2X: Aggiungere 11 g di urea a 10 ml di miscela di gel loading. Mescolare su una piastra calda fino urea si scioglie. Aliquotare in 2 provette ml e conservare a 4 ° C. PAGINA 10 volte mix: Mix 420,4 g di urea, 100 ml di TBE 10X (autoclave), 250 ml di 40% 29:1 Bis / acrilammide in DDH 2 O. Mescolare fino a quando completamente sciolto e regolare il volume a 1 litro. Conservare in bottiglie scure a 4 ° C. TBE 10X: sciogliere 108 g di TRIS, 55 g di acido borico e 9,3 g di EDTA in 1 litro di DDH 2 O. Autoclave e conservare a temperatura ambiente. Tampone di eluizione: Diluire 8 ml di NaCl 5 M, 2 ml di 1 M Tris pH 7,5, 0,4 ml di 0,5 M EDTA pH 8 su 200 ml di DDH 2 O. Autoclave e conservare a temperatura ambiente. </td></tr></tbody></table&gt;

iterative optimization of dna duplexes for crystallization of seqa-dna complexes

Escherichia coli Seqa è un regolatore negativo della replicazione del DNA che impedisce prematuri eventi reinitiation sequestrando cluster hemimethylated GATC entro l&#39;origine di replicazione 1. Al di là della provenienza, Seqa si trova a le forche di replicazione, dove organizza DNA appena replicato in un aumento delle strutture ordinate 2. Associa Seqa solo debolmente con singole sequenze GATC, ma forma complessi ad alta affinità con duplex di DNA contenenti più siti GATC. L&#39;unità minima funzionale e strutturale di Seqa è un dimero, spiegando così il requisito di almeno due sequenze GATC per formare un complesso ad alta affinità con DNA hemimethylated 3. Inoltre, l&#39;architettura Seqa, con l&#39;oligomerizzazione e DNA-legame domini separati da un linker flessibile, consente il collegamento di ripetizioni GATC separati da un massimo di tre spire elicoidali. Pertanto, comprendere la funzione di Seqa a livello molecolare richiede strutturale analisi di Seqa legata a più sequenze GATC. In proteina-DNA cristallizzazione, DNA può avere nessuno ad un eccezionale effetto sulle interazioni di imballaggio a seconda delle dimensioni relative e l&#39;architettura della proteina e del DNA. Se la proteina è più grande della DNA o impronte maggior parte del DNA, l&#39;imballaggio cristallo è mediata principalmente da interazioni proteina-proteina. Viceversa, quando la proteina è la stessa dimensione o minore del DNA o copre solo una frazione delle interazioni DNA, DNA-DNA e DNA-proteina dominano imballaggio cristallo. Pertanto, la cristallizzazione della proteina-DNA complessi richiede lo screening sistematico di 4 DNA lunghezza e termina DNA (smussato o sporgenza) 5-7. In questo rapporto, descriviamo come progettare, ottimizzare, purificare e cristallizzare duplex di DNA contenenti hemimethylated tandem ripete GATC in complesso con una variante dimerica di Seqa (SeqAΔ (41-59)-A25R) per ottenere cristalli adatti per la determinazione della struttura.

Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Struttura cristallina di proteine-DNA può fornire indicazioni in funzione della proteina, meccanismo, così come la natura dell&#39;interazione specifica. Qui, segnaliamo come ottimizzare la lunghezza, la sequenza e le estremità del DNA duplex per la co-cristallizzazione con<em&gt; Escherichia coli</em&gt; Seqa, un regolatore negativo dell&#39;inizio della replicazione.

Ottimizzazione iterativa di duplex di DNA per cristallizzazione di Seqa-DNA Complessi

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

iterative-optimization-dna-duplexes-for-crystallization-seqa-dna

Research

JoVE Journal

Biology

9.9K Views.  McMaster University. Struttura cristallina di proteine-DNA può fornire indicazioni in funzione della proteina, meccanismo, così come la natura dell'interazione specifica. Qui, segnaliamo come ottimizzare la lunghezza, la sequenza e le estremità del DNA duplex per la co-cristallizzazione con Escherichia coli Seqa, un regolatore negativo dell'inizio della replicazione.

Video: Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Cristallizzazione delle proteine ​​per la cristallografia a raggi X

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Ricerca

Biologia

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Cristallizzazione delle proteine ​​di membrana lipidica in Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Cristallizzazione high-throughput di proteine ​​di membrana con il metodo lipidica Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

L&#39;utilizzo di un robot per High-throughput cristallizzazione delle proteine ​​di membrana in mesofasi lipidici

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Ottimizzazione e utilizzo di<em&gt; Agrobacterium</em&gt;-Mediata transitoria produzione di proteine ​​in<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Mapping basato AFM delle proprietà elastiche delle pareti cellulari: al Tissue, cellulare, e le risoluzioni subcellulari

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generazione e Purificazione di umani INO80 rimodellamento della cromatina Complessi e Sottocomplessi

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Efficiente Espressione cellulari di mammifero e Passo singolo Purificazione di extracellulare glicoproteine ​​per cristallizzazione

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificazione, sequenziamento di prossima generazione, e DNA genomico mappatura dei siti di integrazione retrovirali

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...