Gli autori vorrebbero riconoscere Chan-Sien Lay per fornire immagini di microcristalli purificati, Daniel Eriksson e Tom Caradoc-Davies per il supporto alla linea MX2 del Synchrotron australiano e Kathryn Flanagan e Andrew Fryga della struttura FlowCore presso la Monash University per la loro Un&#39;assistenza inestimabile.

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</ol>

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Questo protocollo fornisce due approcci per analizzare i microcristalli allo scopo di facilitare l&#39;analisi di cristalli molto piccoli che sarebbero stati trascurati in passato. Fasi critiche per la purificazione microcristallina Il protocollo presentato è stato ottimizzato utilizzando il polyhedrin Bombyx mori CPV1 espresso in cellule Sf9 come sistema di modello. Tuttavia, i microcristalli in vivo presentano una grande variabilità nella resistenza meccanica. Ad esempio, i cristalli ago-cathepsina B coltivati ​​nelle cellule insettuali sono rigidi e altamente resistenti a sollecitazioni meccaniche e possono essere purificate usando un protocollo simile a quello descritto qui 14 . D&#39;altra parte, i cristalli luciferasi a fiamma, anche coltivati ​​in cellule insettiche coltivate e con una simile morfologia ad ago, si dissolvono immediatamente sulla lisi cellulare 38 . Così, il protocollo di estrazione e purificazione di in vivoI cristalli dovranno essere adattati in una base di errori di prova per casi particolari come descritto nei passaggi 1.3, 2a.3-4, 2b.4, 3.1.3-4 e 3.2a.5. Per i cristalli fragili, la separazione (parziale) dai detriti cellulari potrebbe essere ottenuta mediante centrifugazione a bassa velocità ( cioè 200 xg) o aggiunta di un cuscino di saccarosio nella parte inferiore del tubo invece di ripetuta sonicazione. Come regola generale, i campioni di cristalli preparati per la raccolta dei dati di diffrazione non hanno bisogno di essere altamente purificati. Pertanto, la purificazione rapida è preferibile per raffreddare i cristalli prima che possano decadere. Applicazione a microcristalli in vitro e in vivo Anche se entrambi i protocolli sono qui illustrati con l&#39;analisi dei cristalli cresciuti in vivo , la metodologia può essere facilmente utilizzata per microcristalli coltivati ​​in vassoi di cristallo da proteine ​​ricombinanti purificate. In questoDovrebbero essere considerate le seguenti modifiche: i) il supporto della maglia può essere utilizzato per raccogliere i microcristalli direttamente dalla goccia di cristallizzazione piuttosto che trasferirli pipettando; Ii) poiché la quantità di cristalli può essere un fattore limitante, occorre prendere particolare attenzione quando si assorbe l&#39;eccesso di liquido dal supporto della maglia per evitare l&#39;eccessiva rimozione dei cristalli; Iii) devono essere testati diversi buffer di crioprotectant, come nella cristallografia convenzionale (vedi riferimento 30 ). D&#39;altra parte, per i cristalli in vivo , è fortemente raccomandato l&#39;approccio cellulare . Poiché le cellule sono più facili da visualizzare e manipolate rispetto ai cristalli purificati, questo approccio è più efficiente in termini di utilizzo di tempo e più accessibile ai ricercatori con poca o nessuna esperienza in microcristallografia. È anche più adatto per cristalli fragili che possono essere influenzati dall&#39;estrazione e pur<html> Ificazione dalla cella dovuta a cambiamenti nell&#39;ambiente chimico, diluizione delle proteine ​​circostanti e danni meccanici. Inoltre, le cellule offrono un ambiente protettivo ai cristalli, alleviando la necessità di aggiungere un crioprotettore. L&#39;emissione del trattamento di crioconservazione è stata precedentemente dimostrata per prevenire l&#39;accumulo di difetti e ha migliorato l&#39;isomorfismo tra singoli cristalli, per la raccolta di dati a temperatura ambiente 40 . Il protocollo cellulare inoltra anche l&#39;esigenza di incubazione in soluzione crioprotettori, consentendo comunque la raccolta dati a temperatura criogenica. Nel complesso, nella raccolta di dati di cellulo ha due vantaggi principali: 1) può produrre dati di migliore qualità e risoluzione più elevata per un determinato periodo di tempo di raggi 20 ; E 2) mantiene la proteina cristallizzata in un ambiente cellulare aumentando le possibilità di mantenere un ligando biologicamente rilevante. Ontent &quot;&gt; Limitazioni della tecnica Una complicazione intrinseca della microcristallografia seriale è l&#39;importante numero di set di dati parziali generati durante la raccolta dei dati. Di conseguenza, l&#39;elaborazione dei dati diventa un processo sempre più lungo. Tuttavia, il flusso di lavoro di base per l&#39;elaborazione dei dati può essere ampiamente automatizzato. Ad esempio, la scelta ottimale dei cristalli isomorfi può essere determinata mediante l&#39;analisi cluster gerarchica 28 , ad esempio realizzata in BLEND 36 . Inoltre, i programmi sviluppati per l&#39;analisi dei laser a elettroni liberi a raggi X (XFEL) possono anche essere adattati per elaborare i dati di microcristallografia seriali raccolti sulle linee di travi 2 di sincrotrone e, in alcuni rivelatori automatici di reti di maglie e di rilevamento di cristalli sono state implementate notevolmente facilitando la Raccolta dati 2 , 41 .

L&#39;uso della cristallografia a raggi X per la determinazione di strutture ad alta risoluzione di macromolecole biologiche ha registrato una costante progressione negli ultimi due decenni. La crescente assunzione di cristallografia a raggi X da ricercatori non esperti esemplifica la democratizzazione di questo approccio in molti settori della scienza della vita 1 . Storicamente, i cristalli con dimensioni inferiori a ~ 10 μm sono state considerate come impegnative, se non inutilizzabili, per la determinazione della struttura. La crescente disponibilità di microfocus dedicate a fonti di radiazione di sincrotrone in tutto il mondo e progressi tecnologici, come lo sviluppo di strumenti per la manipolazione dei microcristalli, hanno rimosso gran parte di queste barriere che hanno ostacolato l&#39;ampio utilizzo della microcristallografia a raggi X. Avanzamenti nella microcristallografia a raggi X seriali 2 , 3 e diffrazione a micro elettronica 4 haVe ha mostrato che l&#39;uso di micro- e nanocristalli per la determinazione della struttura non è solo possibile ma anche a volte preferibile all&#39;uso di grandi cristalli 5 , 6 , 7 . Questi progressi sono stati applicati per lo studio dei peptidi 8 e dei cristalli naturali prodotti dai virus degli insetti 9 , 10 . Sono ora utilizzati per una vasta gamma di macromolecole biologiche tra cui i sistemi più difficili come le proteine ​​a membrana e grandi complessi 11 . Per facilitare l&#39;analisi di questi microcristalli, sono stati analizzati in meso, proteine di membrana 12 in particolare e in chips microfluidica 13. La disponibilità di queste metodologie microcristallografia nuovi ha sollevato la possibilità di utilizzare inCristallizzazione vivo come un nuovo percorso per la biologia strutturale 14 , 15 , 16 che offre un&#39;alternativa alla cristallogenesi classica in vitro . Purtroppo, anche quando si possono produrre cristalli in vivo , rimangono diversi ostacoli quali la degradazione o la perdita di ligandi durante la depurazione dalle cellule, la difficoltà nella manipolazione e la visualizzazione dei cristalli alla radiazione di sincrotrone e negli esperimenti di diffusione a raggi X. Come cristalli alternativi sono stati analizzati anche direttamente all&#39;interno della cella senza alcuna fase di purificazione 17 , 18 , 19 . Un&#39;analisi comparativa suggerisce che tali approcci cellulari possono essere più efficienti dell&#39;analisi dei cristalli purificati e dei dati di resa di maggiore risoluzione 20 . Questo protocollo è inTendeva ad aiutare i ricercatori alla microcristallografia delle proteine. Fornisce metodologie che si concentrano sulla preparazione e manipolazione del campione per gli esperimenti di diffrazione a raggi X in un beamline di sincrotrone. Si propongono due opzioni utilizzando cristalli isolati per microcristallografia classica o cellule contenenti cristalli ordinate per citometria a flusso per l&#39;analisi cellulare ( Figura 1 ).

<table><tbody><tr><td>Sf9 cells</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>SF900-SFM insect medium</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>1 L cell culture flask</td><td>Thermofisher Scientific</td><td></td><td></td></tr><tr><td>Shaking incubator for insect cell culture</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>50 mL conical tubes</td><td>Falcon</td><td></td><td></td></tr><tr><td>Centrifuge with swing buckets for 50 mL tubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Sonicator equiped with a 19 mm probe</td><td>MSE Soniprep 150&amp;nbsp;</td><td></td><td></td></tr><tr><td>Glass slides</td><td>Hampton Research</td><td></td><td></td></tr><tr><td>Hemacytometer</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Propidium iodide</td><td>Thermofisher Scientific</td><td></td><td></td></tr><tr><td>BD Influx cell sorter&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Hampton Heavy atom screens</td><td>Hampton Research</td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Micromesh</td><td>Mitigen</td><td></td><td>700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td></tr><tr><td>Paper wick</td><td>Mitigen</td><td></td><td>The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td></tr><tr><td>Ethylene glycol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Trypan blue</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>MX2 microfocus beamline</td><td>Australian Synchrotron</td><td></td><td>A list of available microfocus beamlines can be found in Boudes &lt;em&gt;et al.&lt;/em&gt; (2014) &lt;em&gt;Reflections on the Many Facets of Protein&lt;br/&gt; Microcrystallography.&lt;/em&gt; Australian Journal of Chemistry 67 (12), 1793&amp;ndash;1806,&lt;br/&gt; doi:10.1071/CH14455.</td></tr></tbody></table>

microcrystallography of protein crystals and in cellulo diffraction

L&#39;avvento di linee di microfocus di alta qualità in molti impianti di sincrotrone ha consentito l&#39;analisi di routine di cristalli inferiori a 10 μm nella loro dimensione più grande, che rappresentavano una sfida. Presentiamo due flussi di lavoro alternativi per la determinazione della struttura di microcristalli proteici mediante cristallografia a raggi X con particolare attenzione ai cristalli in vivo . I microcristalli vengono estratti dalle cellule per ultrasuoni e purificati mediante centrifugazione differenziale o analizzati in cellulo dopo la selezione delle cellule mediante citometria a flusso di cellule contenenti cristalli. Opzionalmente, cristalli purificati o cellule contenenti cristalli vengono imbevuti in soluzioni di atomi pesanti per la fase di sperimentazione. Questi campioni vengono quindi preparati per esperimenti di diffrazione in modo analogo mediante applicazione su un supporto micromesh e raffreddamento a flash in azoto liquido. Breve descrizione e confronto di esperimenti di diffrazione seriale di microcristalli isolati e di cristallo-Contenenti le cellule utilizzando un beamline di sincrotrino microfocus per produrre set di dati adatti per la fase di fasi, la costruzione di modelli e la raffinatezza. Questi flussi di lavoro sono esemplificati con i cristalli del polyhedrin Bombyx mori cypovirus 1 (BmCPV1) prodotto dall&#39;infezione di cellule insettiche con un baculovirus ricombinante. In questo caso, l&#39;analisi cellulare è più efficace dell&#39;analisi dei cristalli purificati e produce una struttura in ~ 8 giorni dall&#39;espressione al raffinamento.

Viene presentata una panoramica di entrambi i metodi alternativi per la determinazione della struttura utilizzando microcristalli in vivo ( Figura 1 ). La polihedra può essere facilmente purificata mediante sonicazione e centrifugazione. A causa della loro densità, formano uno strato nella parte inferiore del tubo sotto uno strato di detriti che possono essere rimossi pipettando ( Figura 3a e 3b ). Il campione viene quindi sottoposto a diversi cicli di sonicazione e lavaggi fino a raggiungere un livello sufficiente di purezza come giudicato dall&#39;aspetto bianco e gessoso del pellet ( Figura 3c ). Per l&#39;approccio in cellulo , il profilo di citometria di flusso di cellule non infette viene confrontato con il profilo di cellule contenenti cristalli e utilizzato per determinare quale popolazione cellulare deve essere selezionata per ordinare le cellule contenenti cristalli lontano daLe altre cellule ( Figura 4 ). In questo esempio, le celle contenenti poliedri hanno una dispersione laterale superiore rispetto alle cellule non infette. Altre differenze nei modelli di dispersione possono essere osservate per altri tipi di cristalli e il gating deve essere modificato di conseguenza. I microcristalli purificati o le cellule contenenti cristalli vengono pipettate su un micromesh ( Figura 5 ). Le cellule colorate con blu di trypan possono essere facilmente visualizzate usando telecamere disponibili alla maggior parte delle linee di sincrotrone ( Figura 5b ), mentre i cristalli purificati sono laboriosi per identificare e allinearsi con il raggio a raggi X a causa della loro piccola dimensione ( Figura 5c ). Quando si raccolgono i dati, è meglio procedere in un modello di griglia al fine di ridurre al minimo le procedure di centraggio e per evitare di esporre due volte la stessa cella o cristallo, o mancanti ( Figura 5a ). Raccolta datiDevono essere trattati con attenzione per tenere conto delle differenze nei limiti di diffrazione, dell&#39;effetto dei danni da radiazioni e di una possibile mancanza di isomorfismo. <img alt="Figura 1" class="xfigimg" src="/files/ftp_upload/55793/55793fig1.jpg" /> Figura 1 : Panoramica di due metodi per la determinazione della struttura usando microcristalli in vivo . I microcristalli vengono prodotti in coltura cellulare mediante sovraespressione della proteina di interesse utilizzando un sistema di espressione ricombinante. In casi specifici, i microcristalli possono anche essere prodotti naturalmente dalle cellule in condizioni particolari. La fila superiore mostra l&#39;approccio classico in cui le cellule sono lisate ei cristalli vengono purificati mediante centrifugazione differenziale. I microcristalli purificati vengono pipettati su un micromesh. Dopo la rimozione del liquido in eccesso, si aggiunge una soluzione di crioprotectant, si rimuove il liquido in eccesso e la maglia viene raffreddata a flash. Per raggi X dif<html> Gli esperimenti di frazione, i cristalli sono accuratamente allineati con il raggio a raggi X, che può essere il passo di limitazione della velocità nella raccolta dei dati. La riga inferiore presenta l&#39;approccio cellulare . Le cellule sono ordinate per citometria a flusso per selezionare specificamente le cellule contenenti cristalli. Le cellule sono macchiate con blu tripanico per facilitare la visualizzazione e diffondere su un micromesh. In questo caso, l&#39;aggiunta di un crioprotectante è facoltativa. Per le esperienze di diffrazione dei raggi X, le cellule sono facilmente visualizzate usando tipiche telecamere disponibili nelle fasce di sincrotrone che facilitano il processo di centraggio. Immagine di sistema automatico di cromatografia liquida a cura di GE Healthcare AB, Uppsala, Svezia. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a> <img alt="figura 2" class="xfigimg" src="/files/ftp_upload/55793/55793fig2.jpg" /> = &quot;Xfig&quot;&gt; Figura 2 : Esempi di microcristalli in vivo . Esempi selezionati di cristalli in vivo: ( a ) le cellule Sf9 con poliedra di cypovirus; ( B ) le cellule LD652 con sferoidi di entomopoxvirus; C ) Bacillus thuringiensis con cristalli Cry3A (freccia); ( D ) le cellule Sf9 con i cristalli Trypanosoma brucei cathepsin B; ( E ) le cellule Sf21 con cristalli calcineurin (frecce); ( F ) monoslocce COS7 con PKA: cristalli Inka1; ( G ) le cellule CHO con cristalli IgG. Riprodotto da 14 , 18 , 29 , 37 , 39 , 42 , con autorizzazione. Barra di scala = 10 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig2large.jpg" target="_blank">Clicca qui per vedere un largo</a>Er di questa figura. <img alt="Tabella 1" src="/files/ftp_upload/55793/55793table1.jpg" /> Tabella 1: Sintesi dei cristalli in vivo cresciuti in cellule coltivate che esprimono la proteina ricombinante. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793table1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Viene presentato un protocollo per la cristallografia a raggi X utilizzando microcristalli di proteine. Sono stati confrontati due esempi che analizzano microcristalli in vivo dopo la purificazione o in cellulo .

Microcristallografia dei cristalli proteici e<em&gt; In Cellulo</em&gt; Diffrazione

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

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Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

9.0K Views.  Monash University.  Viene presentato un protocollo per la cristallografia a raggi X utilizzando microcristalli di proteine. Sono stati confrontati due esempi che analizzano microcristalli in vivo dopo la purificazione o in cellulo . 

Video: Microcrystallography of Protein Crystals and In Cellulo Diffraction

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

Cristalli della proteina crescente con dimensioni distinte utilizzando automatizzato cristallizzazione accoppiato con In Situ dispersione della luce dinamica

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Ricerca

Biochimica

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

Chip microfluidici per dispersione della luce dinamica In Situ diffrazione di raggi x di cristallo ed In Situ per la cristallografia seriale

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Chimica

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

Cristallizzazione e determinazione strutturale di un enzima:complesso di substrati mediante cristallografia seriale in un chip microfluidico versatile

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Microcrystal Electron Diffraction of Small Molecules

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

Diffrazione elettronica microcristallina di piccole molecole

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

Cristallizzazione delle proteine su chip mediante microdialisi per studi di diffrazione a raggi X in situ

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Valutazione bidimensionale Prove di cristallizzazione delle proteine ​​di membrana per piccoli studi di biologia strutturale di Cristallografia Electron

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Biologia

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

Cristallizzazione su chip e diffrazione seriale su larga scala a temperatura ambiente

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

Cristallizzazione delle proteine ad alto rendimento tramite microdialisi

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

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 ...

Protein Crystallization

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and computation of an electron density model.
This video introduces protein crystallization and shows a general procedure. Protein expression and purification, crystallization, and x-ray diffraction are covered in the procedure. Applications of protein crystallization include in silico drug design, binding site determination, and membrane protein structure analysis.
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Protein crystallization is the process of obtaining a latticed solid form of a protein. These crystals are especially valuable to structural biologists, assisting in the study of protein function. Other techniques, such as mass spec or SDS-PAGE, can only provide information on the one-dimensional structure of proteins. Protein crystallization is complemented by the techniques of recombinant protein expression and x-ray diffraction. This video will show the principles of protein crystallization, a general laboratory procedure, and several of its applications in the biochemical field.
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The first step required in the process is to obtain milligram quantities of very pure protein, typically using recombinant protein expression. The gene corresponding to the protein of interest is cloned into an expression vector, and the expressed protein is fused to an affinity tag, such as poly-histidine, to assist in the purification by affinity chromatography. To learn more, see this collection&#39;s video on affinity chromatography.
Formation of the purified protein into crystals is dependent on the proper combination of many factors, including pH, ionic strength, concentrations of precipitant and protein, temperature, and rate of equilibration. The most common method used is vapor diffusion, of which there are two categories: hanging drop and sitting drop. A droplet containing pure protein, buffer, and precipitant, which is an ionic solid that binds water molecules, reducing water availability for the protein and mimicking higher protein concentration, is in an enclosed microwell with a reservoir with a more highly concentrated mixture of the same buffer and precipitant. At the beginning, the concentrations of protein and precipitant are too low to cause crystallization. During the course of the experiment, water vaporizes from the droplet and collects in the reservoir; a decrease in the amount of water in the droplet causes the system to become supersaturated, and nucleation, followed by crystallization, can occur. The net transfer of water from the droplet is in equilibrium, and the system is maintained until the process is complete.
To visualize the 3D structure, x-ray diffraction is used. To obtain x-ray data from a crystal, it is placed in a monochromatic x-ray beam, where it is exposed to the beam at all angles. Each exposure provides an image, where each spot is a diffracted x-ray, which emerges from the crystal and is registered by a detector. The data are combined to produce a model of the arrangement of atoms within the crystal. The resulting crystal structure demonstrates the 3-dimensional placement of the atoms, with a typical resolution of 2 angstroms.

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Now that we have covered the principles of protein crystallization, let us look at a generalized protocol.
To begin the procedure an expression vector containing the gene of interest is transformed into cells. The cells are incubated and at mid-log phase, expression is initiated by adding an inducer, such as IPTG, which triggers transcription of the gene&#39;s mRNA. After protein expression, the crude material is suspended in lysis buffer, and then clarified by centrifugation.
The clarified lysate is then loaded onto a nickel column, and the polyhistidine-tagged protein binds to the column while all other biomolecules are washed away.
Once several milligrams of pure protein have been obtained, it is ready for crystallization by vapor diffusion. A 24-well hanging/sitting drop tray is filled with varying concentrations of sodium chloride and sodium acetate buffer solutions. For the sitting drop method, equal volumes of protein and reservoir solution are pipetted onto the shelf above each well, and then the tray is covered with transparent tape. The tray is then placed in an incubation chamber, and the wells are monitored for growth the following day, then every few days.
Once a proper crystal has been obtained it is ready for x-ray diffraction analysis. The crystal is mounted on a goniometer to position the crystal at selected orientations. The crystal is illuminated with a monochromatic beam of x-rays at all angles, producing a diffraction pattern. The software converts the two-dimensional images, taken at different orientations, to a three-dimensional model of the density of electrons within the crystal by determining the positions of the atoms in the crystal.
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Now that we have reviewed a procedure, let&#39;s review some useful applications of protein crystallization, and another crystallization technique.
Protein crystallization may be used for in silico drug design. The three-dimensional structure of Influenza virus&#39;s polymerase basic protein 2, which has been linked to viral infection in mammals, was determined by crystallization and x-ray diffraction. Potential binding sites in the protein are visualized, and with the use of a docking program, a three-dimensional molecule was designed that would insert into a cleft in the protein.
Co-crystallization of protein-DNA complexes is also a useful technique. DNA-binding proteins modulate a wide variety of biological functions such as transcription and DNA polymerization and DNA repair; and crystal structures of these complexes can provide insight into protein function, mechanism, and the nature of the specific interaction. The E. coli protein SeqA, a negative regulator of DNA replication, was co-crystallized with hemimethylated DNA.
Integral membrane proteins such as G-protein coupled receptors, or GCPRs, are difficult to crystallize due to their limited amount of polar surface area available for forming crystal lattice contacts, which has led to the development of fusion-protein-assisted protein crystallization. Genes encoding &#946;2 adrenergic receptor, a GCPR, and a lysozyme were inserted into an expression vector. The crystallization of the &#946;2AR- lysozyme fusion protein was achieved due to the increased extracellular hydrophilic surface over the naturally hydrophobic &#946;2AR, provided by the lysozyme, necessary for forming packing interactions in the crystal lattice.
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You&#39;ve just watched JoVE&#39;s video on protein crystallization. This video described its principles, a generalized protocol, and some its uses in the biomedical field. Thanks for watching!
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