Name: production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad
Uploaded: 2016-12-30T15:00:00.000+00:00
Duration: 13 min 34 s
Description: Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

我们感谢员工同步数据采集期间在瑞士光源，保罗谢勒研究所，菲利根，瑞士支持束线X06DA。我们感谢莫妮卡Gompert优秀的技术支持。该项目由科隆大学，并授予由德国研究委员会INST 216 / 682-1 FUGG支持。从国际研究生院在发展健康和疾病给CP一个博士研究生奖学金是公认的。导致这些结果的研究号赠款协议283570（BioStruct-X）下收到的资金来自欧盟第七框架计划（FP7 / 2007-2013）。

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The authors have nothing to disclose.

X射线晶体学仍然是确定的蛋白质28三维近原子分辨率的结构最快，最准确的方法。但是，它需要秩序井然单晶的生长。这些往往难以得到与结晶状态是人为的。然而，通过X射线晶体与用其他方法，测定确定蛋白质结构的比较特别NMR，通常显示出非常良好的一致性。在PPEP-1的情况下，一个NMR结构最近发表29示出良好的协议与我们的晶体结构14，包括S-环路的流动性。 <p cla...

难辨梭状芽孢杆菌是院内抗生素相关性腹泻感染1的主要原因之一。这种革兰氏阳性厌氧细菌通过其孢子形式通过粪 - 口途径传播。在过去的十年，新的&#39;流行&#39;&#39;或&#39;&#39;超毒&#39;株（ 如 BI / NAP1 / 027）造成的在北美和欧洲的2个新的感染和死亡率急剧增加。 艰难梭菌病机相关性（CDAD）是一种威胁生命结肠炎的高死亡率3。症状的范围从腹泻4至伪膜性肠炎5和经常致命的中毒性巨结肠6。 CDAD治疗是困难的，因为该毒株是耐多药，复发率高7。此刻疗法包括抗生素甲硝唑，fidaxomicin或万古霉素，或者在repetitively复发病例粪便菌群移植。新的治疗策略，迫切需要8。一些进展记录为治疗性单克隆抗体Bezlotoxumab，瞄准艰难梭菌毒素B 9，日前顺利通过III期临床试验，并申请了与FDA和EMA的批准。此外，新的抗生素被此刻测试在临床试验10的不同阶段。 要制定有效的治疗新的治疗靶点必须进行标识。最近发现的艰难梭菌蛋白酶脯氨酸脯氨酸内肽酶-1（PPEP-1; CD2830 / Zmp1; EC 3.4.24.89）是这样一种有前途的靶，如在敲除菌株缺乏PPEP-1的降低的C毒力。难辨体内 11。 PPEP-1是分泌金属蛋白酶12,13在它们的C-末端裂解2 梭状粘附13从而释放粘合杆菌IA从人体肠道上皮细胞。因此，参与维持艰难梭菌的固着和能动表型之间的平衡。为了开发选择性抑制剂对PPEP-1，了解它是如何认识它的底物的三维结构的深入了解是必不可少的。我们已经解决PPEP-1单独和复合物的第一晶体结构的衬底的肽14。 PPEP-1是第一个已知的蛋白酶的两个脯氨酸残基的15之间选择性裂解肽键。其结合在双扭结方式在基片和通过位于在S环覆盖蛋白酶活性位点14个残基的延伸脂族-芳族的网络稳定它。此底物结合模式是唯一的PPEP-1和人蛋白酶迄今未找到。这使得它有前途的药物靶标，以及脱靶酶抑制剂不太可能的影响。 为了发展和屏幕选择PPEP-1 INHibitors在将来需要大量的纯净，单分散PPEP-1蛋白。此外，为了确定与PPEP-1第一抑制剂，共晶体结构的结合将要确定的模式。在我们的手中PPEP-1不断推出共生晶体。因此，我们已开发出一种优化过程，以产生PPEP-1的单衍射质量的晶体。在这个协议中，我们详细描述了PPEP-1 14的生产，纯化，结晶和结构的解决方案。我们在缺乏分泌信号序列，亲和层析和大小排阻色谱法除去纯化标记的一个PPEP-1变体的大肠杆菌用胞内表达，随后通过锌单波长异常色散microseeding 16成优化屏幕和结构确定（锌- SAD）17。这个协议可以适于生产和结构确定的其他蛋白质（ 例如&lt;/ em&gt;的金属蛋白酶），特别是用于生产共生晶体蛋白质。根据要求，构建体的质粒DNA（质粒pET28a-NHIS-rPPEP-1）和衍射数据可以提供用于教育目的。

<table><tbody><tr><td>&lt;strong&gt;Genes / Vectors / cell strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET28a vector</td><td>Merck-Millipore</td><td>69864</td><td>Thrombin cleavable N-terminal His-tag</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; strain BL21 (DE3) Star</td><td>ThermoFisher Scientific</td><td>C601003</td><td>RNase H deficient</td></tr><tr><td>Codon-optimized gene (for &lt;em&gt;E. coli&lt;/em&gt;) of PPEP-1 (CD630_28300)</td><td>Geneart (Thermo Fisher Scientific)</td><td>custom</td><td>amino acids 27-220</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Yeast extract</td><td>any</td><td></td><td></td></tr><tr><td>Tryptone</td><td>any</td><td></td><td></td></tr><tr><td>Antifoam B</td><td>Sigma-Aldrich</td><td>A5757</td><td>aqueous-silicone emulsion</td></tr><tr><td>Agar</td><td>any</td><td></td><td></td></tr><tr><td>Kanamycin</td><td>any</td><td></td><td></td></tr><tr><td>IPTG</td><td>AppliChem</td><td>A1008</td><td></td></tr><tr><td>Tris-HCl</td><td>AppliChem</td><td>A1087</td><td>Buffer grade</td></tr><tr><td>NaCl</td><td>any</td><td></td><td>Buffer grade</td></tr><tr><td>DNaseI</td><td>AppliChem</td><td>A3778</td><td></td></tr><tr><td>Imidazole</td><td>AppliChem</td><td>A1073</td><td>Buffer grade</td></tr><tr><td>Thrombin</td><td>Sigma-Aldrich</td><td>T4648</td><td></td></tr><tr><td>Ammonium phosphate dibasic</td><td>Sigma-Aldrich</td><td>215996</td><td></td></tr><tr><td>Glycerol 100%</td><td>any</td><td></td><td>purest grade</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr><tr><td>Liquid nitrogen</td><td>any</td><td></td><td>for storage and cryocooling of crystals</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment (general)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shaking incubator</td><td>any</td><td></td><td>providing temperatures of 20 &amp;deg;C - 37 &amp;deg;C</td></tr><tr><td>Glassware</td><td>any</td><td></td><td>baffled Erlenmeyer flasks (50 ml - 2.8 L)</td></tr><tr><td>Centrifuge for large culture volumes</td><td>any</td><td></td><td>centrifuge for processing volumes up to 12 L</td></tr><tr><td>Sonicator Vibra-Cell VCX500</td><td>Sonics</td><td>SO-VCX500</td><td>or any other sonicator / cell disruptor</td></tr><tr><td>Ultracentrifuge</td><td>any</td><td></td><td>centrifuge providing speeds up to 150,000 x g</td></tr><tr><td>NiNTA Superflow resin</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Empty Glass Econo-Column</td><td>Bio-Rad</td><td>7371007</td><td>or any other empty glass or plastic column</td></tr><tr><td>Size exclusion chromatography column HiLoad Superdex 200 16/600</td><td>GE Healthcare</td><td>28989335</td><td></td></tr><tr><td>Chromatography system &amp;Auml;kta Purifier</td><td>GE Healthcare</td><td>28406264</td><td>or any other chromatography system</td></tr><tr><td>Dialysis tubing Spectra/Por 3</td><td>Spectrum Labs</td><td>132724</td><td></td></tr><tr><td>Dialysis tubing closures</td><td>Spectrum Labs</td><td>132738</td><td></td></tr><tr><td>Ultrafiltration units (concentrators) 10,000 NWCO</td><td>any</td><td></td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>any</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment (crystallography)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Low volume pipette 0.1-10 &amp;micro;l</td><td>any</td><td></td><td></td></tr><tr><td>Positive displacement pipette Microman M10</td><td>Gilson</td><td>F148501</td><td></td></tr><tr><td>Crystallization robot</td><td>any</td><td></td><td></td></tr><tr><td>96-well crystallization plates TTP IQ with three protein wells</td><td>TTP</td><td>4150-05810</td><td>or any other 96-well crystallization plate&amp;nbsp;</td></tr><tr><td>24-well CombiClover Junior Plate</td><td>Jena Bioscience</td><td>EB-CJR</td><td></td></tr><tr><td>Crystal Clear Sealing Tape</td><td>Hampton Research</td><td>HR3-511</td><td></td></tr><tr><td>Siliconized Glass Cover Slides</td><td>Hampton Research</td><td>HR3-225</td><td></td></tr><tr><td>Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal</td><td>Hampton Research</td><td>diverse</td><td></td></tr><tr><td>Commercial crystallization screens: Wizard, PACT++, JCSG++</td><td>Jena Bioscience</td><td>diverse</td><td></td></tr><tr><td>JBS Beads-for-Seeds</td><td>Jena Bioscience</td><td>CO-501</td><td></td></tr><tr><td>CrystalCap SPINE HT (nylon loops)</td><td>Hampton Research</td><td>diverse</td><td>loop sizes 0.025 mm - 0.5 mm</td></tr><tr><td>CrystalCap Vial</td><td>Hampton Research</td><td>HR4-904</td><td></td></tr><tr><td>Cryogenic Foam Dewar 800 ml</td><td>Hampton Research</td><td>HR4-673</td><td></td></tr><tr><td>Cryogenic Foam Dewar 2 L</td><td>Hampton Research</td><td>HR4-675</td><td></td></tr><tr><td>Vial Clamp, Straight</td><td>Hampton Research</td><td>HR4-670</td><td></td></tr><tr><td>CrystalWand Magnetic, Straight</td><td>Hampton Research</td><td>HR4-729</td><td></td></tr><tr><td>CryoCane 6 Vial Holder</td><td>Hampton Research</td><td>HR4-711</td><td></td></tr><tr><td>CryoSleeve</td><td>Hampton Research</td><td>HR4-708</td><td></td></tr><tr><td>CryoCane Color Coder - White</td><td>Hampton Research</td><td>HR4-713</td><td></td></tr><tr><td>Scalpel</td><td>any</td><td></td><td></td></tr><tr><td>Straight microforcep</td><td>any</td><td></td><td>for manipulation of sealing tape. &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>Acupuncture needle</td><td>any</td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; from a pharmacy</td></tr><tr><td>Stereo microscope</td><td>any</td><td></td><td>for inspection of crystallization plates and crystal mounting, magnification up to 160X</td></tr></tbody></table>

production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

rPPEP-1过表达的几个大肠杆菌菌株，在大肠杆菌 BL21（DE3）中星（ 图1C）的最高产率。第一NiNTA亲和层析步骤之后的6xHis标签可以从大多数蛋白质的成功切除和在第二NiNTA步骤未消化的蛋白质可以从凝血酶消化蛋白质（ 图1D）完全分离。上的S200六百分之十六柱未标记rPPEP-1迁移与偶尔面向最有可能对应于二聚体物种（ 图2A）?...

Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

生产，结晶和结构测定<em&gt;℃。难辨</em&gt;通过Microseeding和锌SAD PPEP-1

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

production-crystallization-structure-determination-c-difficile-ppep-1

Research

JoVE Journal

Biochemistry

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

11.5K 次观看.  University of Cologne. Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein. 

视频: Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

生产二硫键稳定的跨膜肽复合物的结构研究

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

科研

化学

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

生物化学

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

免疫与感染

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

优化用于连续晶体学实验的内硫肽晶体的生长

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

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.

酶的结晶和结构确定:多功能微流体芯片中串行晶体学的基板复合物

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

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

生物学

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.

在油脂介孔膜蛋白的结晶

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

生产，结晶和结构测定

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