Methods for generating peptide libraries and their use as interactive domains in antigen recognition
抗体噬菌体展示技术ppt课件
Meiling Xiong 20180629
1 精选课件PPT
Contents
2
▪ Introduction of Ab phage Display Technology ▪ Ab Formats for Phage Display ▪ Ab Libraries Construction ▪ Phage Ab Selection Methods & Strategies ▪ Phage Ab Screening Applications ▪ In vitro Affinity Maturation ▪ Expression & Purification of Phage Ab Fragments
6
▪ More efficiently than through conventional hybridoma system.
▪ Cheaper to produce recombinant antibodies using bacteria,
rather than mammalian cell line.
protein analysis
▪ Restriction enzyme recognition sites: useful for DNA
recombination and gene manipulation; multiple cloning sites (MCS)
▪ Coat protein: PIII (larger protein, less than 5 copies,)
▪ Nonlytic filamentous phage is the most
trypsin活性测量方法
Trypsin 背景介绍与实验设计Trypsin, introduction form sigma/life-science/metabolomics/enzyme-explorer/analytical-enzymes/trypsin.htmlPhysical Properties and In Vivo ProcessingTrypsinEnzyme Commission (EC) Number: 3.4.21.4Molecular Weight: 23.3 kDa1,2 (bovine & porcine)Extinction Coefficient: E1% = 12.9 - 15.4 (280 nm)3,4pI: 10.1 - 10.52,5(bovine)TrypsinogenMolecular Weight: 24 kDa5 (bovine)Extinction Coefficient: E1% = 14.4 (280 nm)pI: 9.32 (bovine)Trypsinogen, the proenzyme (zymogen) form of trypsin, is produced in the acinar exocrine cells of the pancreas. Three isoforms are excreted from the human pancreas. The cationic and anionic forms are the predominant human isoforms. The inhibitor-resistant mesotrypsinogen is found only in trace amounts.6 The proenzyme is activated only after it reaches the lumen of the small intestine. Enterokinase activates pancreatic trypsinogen to trypsin by the hydrolysis of a hexapeptide(for bovine trypsin at the Lys6 - Ile7 peptide bond) from the NH2 terminus. Bovine trypsinogen consists of a single polypeptide chain of 229 amino acids and is cross linked by six disulfide bridges. Trypsin can autocatalytically activate more trypsinogen to trypsin. Trypsin consists of a single chain polypeptide of 223 amino acid residues. This native form of trypsin is refered to as β-trypsin. Autolysis of β-trypsin (which is cleaved at Lys131- Ser132in the bovine sequence) results in α-trypsin which is held together by disulfide bridges. Trypsin is a member of the serine protease S1 family. The active site amino acid residues of trypsin include His46 and Ser183.2-5Specificity, Kinetics, Substrates and Assay MethodsSpecificity and KineticsTrypsin will cleave peptides on the C-terminal side of lysine and arginine amino acid residues. The rate of hydrolysis is slower if an acidic residue is on either side of the cleavage site and no cleavage occurs if a proline residue is on the carboxyl side of the cleavage site.Trypsin will hydrolyze ester and amide linkages of several synthetic substrates: 2,7,8The pH optimum of trypsin is 7 - 9.10Assay Method11The activity of most Sigma preparations is determined by a continuous rate spectrophotometric assay and expressed in BAEE units.Unit Definition: One BAEE unit will produce a ΔA253 of 0.001 per min at pH 7.6 at 25 °C using BAEE as substrate. Reaction volume = 3.2 mL (1 cm light path).Conditions: Temp = 25 °C, pH = 7.6, A253nm, Light path = 1 cmIn a 3.2 ml reaction mix, the final concentrations are 63 mM sodium phosphate, 0.23 mM Nα-benzoyl-L-arginine ethyl ester, 0.06 mM hydrochloric acid, and 100 units trypsin.Reagents:a. 67 mM Sodium Phosphate Buffer, pH 7.6 at 25 °C (Prepare 100 ml in deionized water using SodiumPhosphate, Monobasic, Anhydrous, Sigma Prod. No. S0751. Adjust to pH 7.6 at 25 °C with 1 M NaOH.)b. 0.25 mM Na-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE) (Prepare 50 ml in Reagent A using Nα-Benzoyl-L-Arginine Ethyl Ester, Hydrochloride, Sigma Prod. No. B4500.)c. 1 mM Hydrochloric Acid Solution (HCl) (Prepare 50 ml in deionized water using concentrated HydrochloricAcid, Sigma Prod. No. 258148.)d. Trypsin Enzyme Solution (Immediately before use, prepare a solution containing 500 BAEE units/ml ofTrypsin in cold Reagent C.)Procedure:Pipette (in milliliters) the following reagents into suitable quartz cuvettes:Test BlankReagent B (BAEE) 3.00 3.00Equilibrate to 25 °C. Monitor the A253nm until constant, using a suitably thermostatted spectrophotometer. Then add:Test BlankReagent C (HCl) --- 0.20Reagent D (Enzyme Solution) 0.20 ---Immediately mix by inversion and record the increase in A253nm for approximately 5 minutes. Obtain theΔA253nm/minute using the maximum linear rate for both the Test and Blank.Calculation:df = Dilution factor0.001 = The change in A253nm/minute per unit of Trypsin at pH 7.6 at 25 °C in a 3.2 ml reaction mix0.20 = Volume (in milliliters) of enzyme usedNotes:1. This assay procedure is not to be used to assay immobilized trypsins2. For the USP/NF procedure refer to the USP monograph3. This procedu re is for informational purposes. For a current copy of Sigma’s quality control procedurecontact our Technical Service Department.Unit Conversions1 BAEE µM Unit = 200 BAEE Units1 TAME µM Unit = 0.27 BAEE µM Units1 BAEE µM Unit = 3.64 TAME Units1 TAME µM Unit = 55 BAEE A253 Units1 BAEE A253 Unit = 0.018 TAME µM Unit1 TAME µM Unit = 180 TAME A247 Units1 TAME A247 Unit = 0.33 BAEE Units1 USP Unit = 3.0 BAEE Units1 NF Unit = 1.1 USP UnitsReconstitution and Solution StabilityTrypsin solutions in 1 mM HCl (pH 3) are stable for approximately 1 year when aliquoted and stored at -20 °C. The presence of Ca2+(20 mM) will also retard trypsin's ability to selfdigest itself (autolysis) and will maintain the stability of the trypsin in solution.2,9 Trypsin retains most of its activity in 2.0 M urea, 2.0 M guanidine HCl, or 0.1% (w/v) SDS.13 Trypsin is reversibly denatured at high pH (above 11), by precipitation with TCA, or by high concentrations of urea (greater than 6.5 M).3 In order to abolish all trypsin activity, heating at 100 °C in 1% (w/v) SDS for 5 minutes is required.14Trypsinogen solutions are stable in acidic buffers (pH 2 - 4), while in neutral buffers the autocatalytic activation to trypsin occurs.References1. Cunningham, L. W. Jr., Molecular Kinetic properties of crystalline diisopropyl phosphoryl trypsin.J. Biol. Chem., 211, 13-19 (1954).2. Walsh, K. A., Trypsinogens and trypsins of various species. Meth. Enzymol., 19, 41-63 (1970).3. Keil, B., in The Enzymes, 3rd ed., Vol. III, Boyer, P. D., Academic Press (New York, NY: 1971),pp. 250-275.4. Shaw, E., et al., Evidence for an active center histidine in trypsin through use of a specificreagent, 1-chloro-3-tosylamido-7-amino-2-heptanone, the chloromethyl ketone derived from Nα-tosyl-L-lysine. Biochemistry, 4(10), 2219-2224 (1965).5. Tietze, F., Molecular kinetic properties of crystalline trypsinogen. J. Biol. Chem., 204(1), 1-11(1953).6. Chen, J-M; Ferec, C., Genes, Cloned cDNAs, and Proteins of Human Trypsinogens andPancreatitis-Associated Cationic Trypsinogen Mutations. Pancreas, 21, 57-62 (2000)7. Burdon, R. H., et al., in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 9,2nd ed., Allen, G., ed., Elsevier/North (New York, NY: 1989), pp. 73-104.8. Enzyme Handbook, Vol. II, Barman, T. E., Springer-Verlag (New York, NY: 1969), pp. 618-619.9. Enzyme Handbook, Vol. II, Barman, T. E., Springer-Verlag (New York, NY: 1969), p. 819.10. Sipos, T., and Merkel, J. R., An effect of calcium ions on the activity, heat stability, and structureof trypsin. Biochemistry, 9(14), 2766-2775 (1970).11. Bergmeyer, H.U., Gawehn, K., and Grassl, M. (1974) in Methods of Enzymatic Analysis(Bergmeyer, H.U., ed) Volume I, 2nd ed., 515-516, Academic Press, Inc.,New York, NY12. Wang, S. S., and Carpenter, F. H., Kinetics of the tryptic hydrolysis of the oxidized B chain ofbovine insulin. Biochemistry, 6(1), 215-224 (1967).13. Carpenter, F. H., Treatment of trypsin with TPCK. Methods Enzymol., 11, 237 (1967).14. Methods of Molecular Biology, Vol. 3, Smith, B. J., Humana Press, (New Jersey, 1988), pp. 57-69.Trypsin, Proteomics Grade from sigma/life-science/proteomics/mass-spectrometry/trypsin-proteomics-grade.html#Trypsin, Proteomics GradeTrypsin, Proteomics GradeSuperior Sequence Coverage Without Ambiguous Peaks!Proteomics Grade Trypsin (T6567) shows higher proteolytic efficiency than competitors' products, generating more tryptic peptides leading to greater sequence coverage of your proteins of interest. Mass spectra are significantly simplified due to the reduced number of interfering autolytic peaks and their ambiguous adducts. (Concerns over the lack of internal calibrant peaks at low mass may be addressed by calibrating on matrix peaks that routinely appear in the mass spectrum under normal circumstances.)Proteomics Grade Trypsin has been extensively purified from porcine pancreas to enable accurate and precise cleavage on the carboxylic acid side of Arg and Lys residues. The enzyme has been exhaustively processed by reductive methylation to minimize autolysis and chymotryptic activity quenched by TPCK (N-Tosyl-L-phenylalanine chloromethyl ketone) treatment. Further purification steps including affinity chromatography and lyophilization from dilute acid produce a highly-purified, high specific activity trypsin purposely suited for the demanding criteria of proteomics research, and is designed to function for either in-solution or in-gel digests. The enzyme is conveniently packaged in five 20 µg vials to ensure fresh enzyme is available for each use.Trypsin-ultra™, Mass Spectrometry Grade (NEB)1 IVGGYTCAEN SVPYQVSLNA GYHFCGGSLI NDQWVVSAAH CYQYHIQVRL GEYNIDVLEG61 GEQFIDASKI IRHPKYSSWT LDNDILLIKL STPAVINARV STLLLPSACA SAGTECLISG121 WGNTLSSGVN YPDLLQCLVA PLLSHADCEA SYPGQITNNM ICAGFLEGGK DSCQGDSGGP181 VACNGQLQGI VSWGYGCAQK GKPGVYTKVC NYVDWIQETI AANSTrypsin-ultra™, Mass Spectrometry Gradei is a serine endopeptidase. It selectively cleaves peptide bonds C-terminal to lysine and arginine residues (1). Trypsin-ultra is treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) to inactivate any remaining chymotryptic activity. It is modified by acetylation of the ε-amino groups of lysine residues to prevent autolysis. Trypsin-ultra (TPCKtreated) cleaves at Lys-Pro and Arg-Pro bonds at a much slower rate than other amino acid residues (2).Product SourceIsolated from bovine (Bos taurus) pancreasReaction Conditions1X Trypsin-ultra, Reaction BufferIncubate at 37°CTrypsin-ultra, Reaction Buffer:50 mM Tris-HCl20 mM CaCl2pH 8 @ 25°CUsage Concentration100ng/μlStorage Temperature-20°CMolecular WeightsTheoretical: 23675 daltonsSpecific Activity2.1 μmol/min/mg/cationic trypsin precursor [Bos taurus](NEB为参考)/protein/2499861?report=genbank&log$=protalign&blast_rank=1&RID=KBS2F7KN016/nucleotide/829?report=genbank&log$=nucltop&blast_rank=1&RID=KBSZCEXR01R1 ATGCATCCCC TGCTTATCCT TGCCTTTGTG GGAGCTGCTG TGGCTTTCCC CTCGGACGAC61 GATGACAAGA TCGTCGGGGG CTACACCTGC GCAGAGAATT CCGTCCCTTA CCAGGTGTCC121 CTGAATGCTG GCTACCACTT CTGCGGGGGC TCCCTCATCA ATGACCAGTG GGTGGTGTCC181 GCGGCTCACT GCTACCAGTA CCACATCCAG GTGAGGCTGG GAGAATACAA CATTGATGTC241 TTGGAGGGTG GTGAGCAGTT CATCGATGCG TCCAAGATCA TCCGCCACCC CAAGTACAGC301 AGCTGGACTC TGGACAATGA CATCCTGCTG ATCAAACTCT CCACGCCTGC GGTCATCAAT361 GCCCGGGTGT CCACCTTGCT GCTGCCCAGT GCCTGTGCTT CCGCAGGCAC AGAGTGCCTC421 ATCTCCGGCT GGGGCAACAC CCTGAGCAGT GGCGTCAACT ACCCGGACCT GCTGCAATGC481 CTGGTGGCCC CGCTGCTGAG CCACGCCGAC TGTGAAGCCT CATACCCTGG ACAGATCACT541 AACAACATGA TCTGCGCTGG CTTCCTGGAA GGAGGCAAGG ATTCCTGCCA GGGTGACTCT601 GGCGGCCCTG TGGCTTGCAA CGGACAGCTC CAGGGCATTG TGTCCTGGGG CTACGGCTGT661 GCCCAGAAGG GCAAGCCTGG GGTCTACACC AAGGTCTGCA ACTACGTGGA CTGGATTCAG721 GAGACCATCG CCGCCAACAG CTGA本实验的实验方向：１、实现表达和纯化；２、固定化trypsin,开发可离心式的酶切工具去掉信号肽以后的表达蛋白序列1 MFPSDDDDK I VGGYTCAENS VPYQVSLNAG YHFCGGSLIN DQWVVSAAHC YQYHIQVRLG61 EYNIDVLEGG EQFIDASKII RHPKYSSWTL DNDILLIKLS TPAVINARVS TLLLPSACAS 121 AGTECLISGW GNTLSSGVNY PDLLQCLVAP LLSHADCEAS YPGQITNNMI CAGFLEGGKD 181 SCQGDSGGPV ACNGQLQGIV SWGYGCAQKG KPGVYTKVCN YVDWIQETIA ANS*去掉信号肽以后的基因序列1 ATG TTCCCCT CGGACGACGA TGACAAGATC GTCGGGGGCT ACACCTGCGC AGAGAATTCC61 GTCCCTTACC AGGTGTCCCT GAATGCTGGC TACCACTTCT GCGGGGGCTC CCTCATCAAT121 GACCAGTGGG TGGTGTCCGC GGCTCACTGC TACCAGTACC ACATCCAGGT GAGGCTGGGA181 GAATACAACA TTGATGTCTT GGAGGGTGGT GAGCAGTTCA TCGATGCGTC CAAGATCATC241 CGCCACCCCA AGTACAGCAG CTGGACTCTG GACAATGACA TCCTGCTGAT CAAACTCTCC301 ACGCCTGCGG TCATCAATGC CCGGGTGTCC ACCTTGCTGC TGCCCAGTGC CTGTGCTTCC361 GCAGGCACAG AGTGCCTCAT CTCCGGCTGG GGCAACACCC TGAGCAGTGG CGTCAACTAC421 CCGGACCTGC TGCAATGCCT GGTGGCCCCG CTGCTGAGCC ACGCCGACTG TGAAGCCTCA481 TACCCTGGAC AGATCACTAA CAACATGATC TGCGCTGGCT TCCTGGAAGG AGGCAAGGAT541 TCCTGCCAGG GTGACTCTGG CGGCCCTGTG GCTTGCAACG GACAGCTCCA GGGCATTGTG601 TCCTGGGGCT ACGGCTGTGC CCAGAAGGGC AAGCCTGGGG TCTACACCAA GGTCTGCAAC661 TACGTGGACT GGATTCAGGA GACCATCGCC GCCAACAGC T GA载体信息： pET11C密码子优化结果：基因合成以及两段的酶切位点信息(Nde I)1 ATG TTCCCCT CGGACGACGA TGACAAGATC GTCGGGGGCT ACACCTGCGC AGAGAATTCC 61 GTCCCTTACC AGGTGTCCCT GAATGCTGGC TACCACTTCT GCGGGGGCTC CCTCATCAAT 121 GACCAGTGGG TGGTGTCCGC GGCTCACTGC TACCAGTACC ACATCCAGGT GAGGCTGGGA 181 GAATACAACA TTGATGTCTT GGAGGGTGGT GAGCAGTTCA TCGATGCGTC CAAGATCATC 241 CGCCACCCCA AGTACAGCAG CTGGACTCTG GACAATGACA TCCTGCTGAT CAAACTCTCC 301 ACGCCTGCGG TCATCAATGC CCGGGTGTCC ACCTTGCTGC TGCCCAGTGC CTGTGCTTCC 361 GCAGGCACAG AGTGCCTCAT CTCCGGCTGG GGCAACACCC TGAGCAGTGG CGTCAACTAC 421 CCGGACCTGC TGCAATGCCT GGTGGCCCCG CTGCTGAGCC ACGCCGACTG TGAAGCCTCA 481 TACCCTGGAC AGATCACTAA CAACATGATC TGCGCTGGCT TCCTGGAAGG AGGCAAGGAT 541 TCCTGCCAGG GTGACTCTGG CGGCCCTGTG GCTTGCAACG GACAGCTCCA GGGCATTGTG 601 TCCTGGGGCT ACGGCTGTGC CCAGAAGGGC AAGCCTGGGG TCTACACCAA GGTCTGCAAC 661 TACGTGGACT GGATTCAGGA GACCATCGCC GCCAACAGC T GA (BamH I)。
FLAG_peptide_SDS_MedChemExpress
Inhibitors, Agonists, Screening LibrariesSafety Data Sheet Revision Date:Sep.-22-2017Print Date:Sep.-22-20171. PRODUCT AND COMPANY IDENTIFICATION1.1 Product identifierProduct name :FLAG peptideCatalog No. :HY-P0223CAS No. :98849-88-81.2 Relevant identified uses of the substance or mixture and uses advised againstIdentified uses :Laboratory chemicals, manufacture of substances.1.3 Details of the supplier of the safety data sheetCompany:MedChemExpress USATel:609-228-6898Fax:609-228-5909E-mail:sales@1.4 Emergency telephone numberEmergency Phone #:609-228-68982. HAZARDS IDENTIFICATION2.1 Classification of the substance or mixtureNot a hazardous substance or mixture.2.2 GHS Label elements, including precautionary statementsNot a hazardous substance or mixture.2.3 Other hazardsNone.3. COMPOSITION/INFORMATION ON INGREDIENTS3.1 SubstancesSynonyms:DYKDDDDK; Asp–Tyr–Lys–Asp–Asp–Asp–Asp–LysFormula:C41H60N10O20Molecular Weight:1012.97CAS No. :98849-88-84. FIRST AID MEASURES4.1 Description of first aid measuresEye contactRemove any contact lenses, locate eye-wash station, and flush eyes immediately with large amounts of water. Separate eyelids with fingers to ensure adequate flushing. Promptly call a physician.Skin contactRinse skin thoroughly with large amounts of water. Remove contaminated clothing and shoes and call a physician.InhalationImmediately relocate self or casualty to fresh air. If breathing is difficult, give cardiopulmonary resuscitation (CPR). Avoid mouth-to-mouth resuscitation.IngestionWash out mouth with water; Do NOT induce vomiting; call a physician.4.2 Most important symptoms and effects, both acute and delayedThe most important known symptoms and effects are described in the labelling (see section 2.2).4.3 Indication of any immediate medical attention and special treatment neededTreat symptomatically.5. FIRE FIGHTING MEASURES5.1 Extinguishing mediaSuitable extinguishing mediaUse water spray, dry chemical, foam, and carbon dioxide fire extinguisher.5.2 Special hazards arising from the substance or mixtureDuring combustion, may emit irritant fumes.5.3 Advice for firefightersWear self-contained breathing apparatus and protective clothing.6. ACCIDENTAL RELEASE MEASURES6.1 Personal precautions, protective equipment and emergency proceduresUse full personal protective equipment. Avoid breathing vapors, mist, dust or gas. Ensure adequate ventilation. Evacuate personnel to safe areas.Refer to protective measures listed in sections 8.6.2 Environmental precautionsTry to prevent further leakage or spillage. Keep the product away from drains or water courses.6.3 Methods and materials for containment and cleaning upAbsorb solutions with finely-powdered liquid-binding material (diatomite, universal binders); Decontaminate surfaces and equipment by scrubbing with alcohol; Dispose of contaminated material according to Section 13.7. HANDLING AND STORAGE7.1 Precautions for safe handlingAvoid inhalation, contact with eyes and skin. Avoid dust and aerosol formation. Use only in areas with appropriate exhaust ventilation.7.2 Conditions for safe storage, including any incompatibilitiesKeep container tightly sealed in cool, well-ventilated area. Keep away from direct sunlight and sources of ignition.Recommended storage temperature:Powder-20°C 3 years4°C 2 yearsIn solvent-80°C 6 months-20°C 1 monthShipping at room temperature if less than 2 weeks.7.3 Specific end use(s)No data available.8. EXPOSURE CONTROLS/PERSONAL PROTECTION8.1 Control parametersComponents with workplace control parametersThis product contains no substances with occupational exposure limit values.8.2 Exposure controlsEngineering controlsEnsure adequate ventilation. Provide accessible safety shower and eye wash station.Personal protective equipmentEye protection Safety goggles with side-shields.Hand protection Protective gloves.Skin and body protection Impervious clothing.Respiratory protection Suitable respirator.Environmental exposure controls Keep the product away from drains, water courses or the soil. Cleanspillages in a safe way as soon as possible.9. PHYSICAL AND CHEMICAL PROPERTIES9.1 Information on basic physical and chemical propertiesAppearance White to off-white (Solid)Odor No data availableOdor threshold No data availablepH No data availableMelting/freezing point No data availableBoiling point/range No data availableFlash point No data availableEvaporation rate No data availableFlammability (solid, gas)No data availableUpper/lower flammability or explosive limits No data availableVapor pressure No data availableVapor density No data availableRelative density No data availableWater Solubility No data availablePartition coefficient No data availableAuto-ignition temperature No data availableDecomposition temperature No data availableViscosity No data availableExplosive properties No data availableOxidizing properties No data available9.2 Other safety informationNo data available.10. STABILITY AND REACTIVITY10.1 ReactivityNo data available.10.2 Chemical stabilityStable under recommended storage conditions.10.3 Possibility of hazardous reactionsNo data available.10.4 Conditions to avoidNo data available.10.5 Incompatible materialsStrong acids/alkalis, strong oxidising/reducing agents.10.6 Hazardous decomposition productsUnder fire conditions, may decompose and emit toxic fumes.Other decomposition products - no data available.11.TOXICOLOGICAL INFORMATION11.1 Information on toxicological effectsAcute toxicityClassified based on available data. For more details, see section 2Skin corrosion/irritationClassified based on available data. For more details, see section 2Serious eye damage/irritationClassified based on available data. For more details, see section 2Respiratory or skin sensitizationClassified based on available data. For more details, see section 2Germ cell mutagenicityClassified based on available data. For more details, see section 2CarcinogenicityIARC: No component of this product present at a level equal to or greater than 0.1% is identified as probable, possible or confirmed human carcinogen by IARC.ACGIH: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by ACGIH.NTP: No component of this product present at a level equal to or greater than 0.1% is identified as a anticipated or confirmed carcinogen by NTP.OSHA: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by OSHA.Reproductive toxicityClassified based on available data. For more details, see section 2Specific target organ toxicity - single exposureClassified based on available data. For more details, see section 2Specific target organ toxicity - repeated exposureClassified based on available data. For more details, see section 2Aspiration hazardClassified based on available data. For more details, see section 212. ECOLOGICAL INFORMATION12.1 ToxicityNo data available.12.2 Persistence and degradabilityNo data available.12.3 Bioaccumlative potentialNo data available.12.4 Mobility in soilNo data available.12.5 Results of PBT and vPvB assessmentPBT/vPvB assessment unavailable as chemical safety assessment not required or not conducted.12.6 Other adverse effectsNo data available.13. DISPOSAL CONSIDERATIONS13.1 Waste treatment methodsProductDispose substance in accordance with prevailing country, federal, state and local regulations.Contaminated packagingConduct recycling or disposal in accordance with prevailing country, federal, state and local regulations.14. TRANSPORT INFORMATIONDOT (US)This substance is considered to be non-hazardous for transport.IMDGThis substance is considered to be non-hazardous for transport.IATAThis substance is considered to be non-hazardous for transport.15. REGULATORY INFORMATIONSARA 302 Components:No chemicals in this material are subject to the reporting requirements of SARA Title III, Section 302.SARA 313 Components:This material does not contain any chemical components with known CAS numbers that exceed the threshold (De Minimis) reporting levels established by SARA Title III, Section 313.SARA 311/312 Hazards:No SARA Hazards.Massachusetts Right To Know Components:No components are subject to the Massachusetts Right to Know Act.Pennsylvania Right To Know Components:No components are subject to the Pennsylvania Right to Know Act.New Jersey Right To Know Components:No components are subject to the New Jersey Right to Know Act.California Prop. 65 Components:This product does not contain any chemicals known to State of California to cause cancer, birth defects, or anyother reproductive harm.16. OTHER INFORMATIONCopyright 2017 MedChemExpress. The above information is correct to the best of our present knowledge but does not purport to be all inclusive and should be used only as a guide. The product is for research use only and for experienced personnel. It must only be handled by suitably qualified experienced scientists in appropriately equipped and authorized facilities. The burden of safe use of this material rests entirely with the user. MedChemExpress disclaims all liability for any damage resulting from handling or from contact with this product.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。
抗体噬菌体展示技术(课堂PPT)
host.
▪ Excellent potential to further improve binding properties of the
selected antibody by protein engineering techniques.
▪ Amber codon TAG: supE strains (glutamic acid
codon), non-suppressor strains (stop codon)
▪ Protease cleavage site
▪ Promoter
▪ Signal peptides: phage protein translocation, crucial
for display level
▪ Selective marker: for selection of infected host cells
5
Introduction of Phage Display Technology
▪ Nonlytic filamentous phage is the most
6
▪ More efficiently than through conventional hybridoma system.
▪ Cheaper to produce recombinant antibodies using bacteria,
rather than mammalian cell line.
polystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and
拉伸模拟umbrella sampling
G ROMACS TutorialUmbrella SamplingJustin LemkulDepartment of Biochemistry,Virginia TechThis tutorial will guide the user through the process of setting up and running pulling simulations necessary to calculate binding energy between two species. The tutorial assumes the user has already successfully completed the Lysozyme tutorial,some other tutorial,or is otherwise well-versed in basic G ROMACS simulation methods and topology organization.Special attention will be paid to the methods for properly building the system and settings for the pull code itself.)is derived from the potential of mean force(PMF), The binding energy(∆Gbindextracted from a series of umbrella sampling simulations.A series of initial configurations is generated,each corresponding to a location wherein the molecule of interest(generally referred to as a"ligand")is harmonically restrained at increasing center-of-mass(COM)distance from a reference molecule using an umbrella biasing potential.This restraint allows the ligand to sample the configurational space in a defined region along a reaction coordinate between it and its reference molecule or binding partner.The windows must allow for slight overlap of the ligand positions for proper reconstruction of the PMF curve. The steps for such a procedure(and the ones utilized in this tutorial)are as follows:1Generate a series of configurations along a single degree of freedom (reaction coordinate)2Extract frames from the trajectory in step1that correspond to the desired COM spacing3Run umbrella sampling simulations on each configuration to restrain it within a window corresponding to the chosen COM distance4Use the Weighted Histogram Analysis Method(WHAM)to extract the PMF andcalculate∆GbindThe tutorial assumes that the reader is using G ROMACS version4.0.5or later,to take advantage of bug fixes pertinent to the pull code.The pull code was completely re-written after version3.3.3,such that none of the information contained herein (beyond the basic theory of the technique)is applicable to any G ROMACS versionprior to 4.0.Start the Tutorial!Tutorials HomeG ROMACS TutorialStep One:Prepare the TopologyGenerating a molecular topology for an umbrella sampling simulation is just like any other simulation.Obtain the coordinate file of the structure of interest, and generate the topology from pdb2gmx.Some systems will require special consideration(i.e.,protein-ligand complexes,membrane proteins,etc).For protein-ligand systems,please consult this tutorial,and for membrane proteins, I recommend my own tutorial on the topic.The principles of umbrella sampling are easily extendable to these systems,though we will consider only protein molecules in this tutorial.The system we will consider here is the dissociation of a single peptide from the growing end of an Aβ42protofibril,and is based on simulations we recentlypublished.The structure file of the wild-type Aβ42protofibril used in those simulations,acetylated at the N-terminus of each chain,can be found here.The original PDB accession code is2BEG.Run the structure through pdb2gmx:pdb2gmx-f input.pdb-ignh-ter-o complex.groChoose the G ROMOS9653A6parameter set,"None"for the N-termini,and"COO-"for the C-termini.Modify topol_B.itp to include the following lines(at the end of the file):#ifdef POSRES_B#include"posre_B.itp"#endifWe will be using chain B as an immobile reference later on in the pulling simulations,hence the need to specially position-restrain this chain only,and none of the others.PLEASE NOTE:Unfortunately,pdb2gmx does not properly generate the CA-C-O angle that must be written to the topology.Without these parameters,grompp will fail later.You have two options to fix this problem:1Add a line for this angle in the ACE.rtp entry:"CA C O ga_30"in the[angles] directive2Manually add"ga_30"to the line in the[angles]directive of topol_*.itp where it is missingBack: Introduction Next:Defining theUnit CellG ROMACS TutorialStep Two:Define the Unit CellDefining the unit cell for a pulling simulation is not unlike defining the unit cell for any other simulation.There is,however,one major consideration.One must allow enough space in the pulling direction to allow for a continuous pull without interacting with the periodic images of the system.That is,the minimum image convention must be continually satisfied,and as well,the pull distance must always be less than one-half the length of the box vector along which the pulling is being conducted.Why,you may ask?G ROMACS calculates distances while simultaneously taking periodicity into account.This,if you have a 10-nm box,and you pull over a distance greater than 5.0nm,the periodic distance becomes the reference distance for the pulling,and this distance is actually less than 5.0nm!This fact will significantly affect results,since the distance you think you are pulling is not what is actually calculated.We will be pulling a total distance of 5.0nm in a 12.0-nm box,to avoid the complications described above.The center of mass of the protofibril will be placed at (3.280,2.181,2.4775)in a box of dimensions 6.560x 4.362x e editconf to place the protofibril at this location:editconf -f complex.gro -o newbox.gro -center 3.2802.1812.4775-box 6.5604.36212You can visualize the location of the protofibril within the box using,for example,VMD.Load the structure in VMD and open the Tk console.Type the following (note that >indicates the Tk prompt,not something you actually type):>package require pbctools>pbc boxYou should see something like the following in the VMD window:Back:TheTopology Next:Solvation and IonsG ROMACS TutorialStep Three:Adding Solvent and IonsThis step is conducted much like any other simulation.Refer to the Lysozyme tutorial for a more detailed description of what is going on here if you are unsure. First,we will add water with genbox:genbox-cp newbox.gro-cs spc216.gro-o solv.gro-p topol.topNext,we will add ions using genion,utilizing this.mdp file.We are going to be conducting these simulations in the presence of100mM NaCl,on top of neutralizing counterions:grompp-f ions.mdp-c solv.gro-p topol.top-o ions.tprgenion-s ions.tpr-o solv_ions.gro-p topol.top-pname NA+-nname CL--neutral -conc0.1Back:Defining the Unit Cell Next:Energy Minimization and EquilibrationG ROMACS TutorialStep Four:Energy Minimization and EquilibrationThe energy minimization and equilibration steps are going to be conducted just like any other protein-in-water system.Here,we will perform steepest descents minimization followed by NPT equilibration.The.mdp file for minimization can be found here,and the one for NPT equilibration can be found here.Invoke grompp and mdrun,as usual:grompp-f minim.mdp-c solv_ions.gro-p topol.top-o em.tprmdrun-v-deffnm emgrompp-f npt.mdp-c em.gro-p topol.top-o npt.tprmdrun-deffnm nptBecause these procedures are time-consuming,they are likely best run on a cluster using MPI-enabled mdrun,i.e.:mpirun-np X mdrun_mpi-deffnm nptIn the above command,"X"represents the desired number of processors over which the parallel calculation is conducted.Back:Adding Solvent and Ions Next:Generating ConfigurationsG ROMACS TutorialStep Five:Generating ConfigurationsTo conduct umbrella sampling,one must generate a series of configurations along a reaction coordinate,ζ.Some of these configurations will serve as the starting configurations for the umbrella sampling windows,which are run in independent simulations.The figure below illustrates these principles.The top image illustrates the pulling simulation we will run now,conducted in order to generate a series of configurations along the reaction coordinate.These configurations are extracted after the simulation is complete(dashed arrows in between the top and middle images).The middle image corresponds to the independent simulations conducted within each sampling window,with the center of mass of the free peptide restrained in that window by an umbrella biasing potential.The bottom images shows the ideal result as a histogram of configurations,with neighboring windows overlapping such that a continuous energy function can later be derived from these simulations.For this example,the reaction coordinate is the z-axis.To generate these configurations,we must pull peptide A away from the protofibril.We will pull over the course of500ps of MD,saving snapshots every1ps.This setup has been established based on trial-and-error to obtain a reasonable distribution of configurations.In other systems,it may be necessary to save configurations more often,or sufficient to save configurations less often.The idea is to capture enough configurations along the reaction coordinate to obtain regular spacing of the umbrella sampling windows,in terms of center-of-mass distance between peptides A and B,the latter of which is our reference group.The.mdp file for this pulling can be found here.A brief explanation of the pulling options used is as follows:;Pull codepull=umbrellapull_geometry=distancepull_dim=N N Ypull_start=yes;define initial COM distance>0pull_ngroups=1pull_group0=Chain_Bpull_group1=Chain_Apull_rate1=0.01;0.01nm per ps=10nm per nspull_k1=1000;kJ mol^-1nm^-2•pull=umbrella:using a harmonic potential to pull.IMPORTANT:This procedure is NOT umbrella sampling.I used a harmonic potential in order to make qualitative observations about the dissociation pathway in this study.The harmonic potential allows the force to vary according to the nature of the interactions of peptide A with peptide B.That is,the force will build up until certain critical interactions are broken.See our paper for details.For the purposes of generating the initial configurations for umbrella sampling,you can actually use any combination of pull settings(pull and pull_geometry),but when it comes time for the actual umbrella sampling(in the next step)you MUST be using pull=umbrella.It is very important that you do not apply extremely fast pulling rates or extremely strong force constants,which can seriously deform elements of your system.Please refer to paper(particularly the Supporting Information)for how we chose to validate the pull rate used.•pull_geometry=distance:see the note the in.mdp file;you can also use position or direction,but changes will have to be made to other pulling parameters.•pull_dim=N N Y:we are pulling only in the z-dimension.Thus,x and y are set to"no"(N)and z is set to"yes"(Y).•pull_start=yes:the initial COM distance is the reference distance for the first frame.This is useful because if we are attempting to pull 5.0nm, converting the initial COM distance to zero(i.e.,pull_start=no)makes this interpretation difficult.•pull_ngroups=1:we are only applying a pulling force to one group.•pull_group0=Chain_B:reference group for pulling.•pull_group1=Chain_A:group to which pulling force is applied.•pull_rate1=0.01:the rate at which the"dummy particle"attached to our pull group is moved.This type of pulling is also called"constant velocity"due to the fact that this rate is fixed.•pull_k1=1000:the force constant for pulling.Remember that#ifdef POSRES_B statement we added to topol_B.itp a while ago?We're going to use it now.By restraining peptide B of the protofibril,we are able to more easily pull peptide A away.Due to the extensive non-covalent interactions between chains A and B,if we did not restrain chain B,we would end up simply towing the whole complex along the simulation box,which wouldn't accomplish much. We will need to define some custom index groups for this pulling e make_ndx:make_ndx-f npt.gro(>indicates the make_ndx prompt)>r1-27>name16Chain_A>r28-54>name17Chain_B>qNow,run the continuous pulling simulation:grompp-f md_pull.mdp-c npt.gro-p topol.top-n index.ndx-t npt.trr-o pull.tpr mdrun-s pull.tprAgain,this procedure will take some time,so run it in parallel if you have the resources available to you.Once this simulation is complete,we will need to extract useful frames for defining the umbrella sampling windows.The easiest way I have found to do this is the following:1Define the spacing of the windows(generally0.1-0.2nm)2Extract all the frames from the pulling trajectory that was just produced 3Measure the COM distance of each of these frames between the reference and pull group4Use the selected frames for umbrella sampling inputTo extract the frames from your trajectory(traj.xtc),use trjconv:trjconv-s pull.tpr-f traj.xtc-o conf.gro-sepA series of coordinate files(conf0.gro,conf1.gro,etc)will be produced, corresponding to each of the frames saved in the continuous pulling simulation. To iteratively call g_dist on all of these(501!)frames that were generated,I have written a Perl script that takes care of this task.It will print a file called "summary_distances.dat"that contains this information.The script can be found here.We will need to make use of the index file again,as well as a text file called"groups.txt,"which will be used to select our analysis groupsnon-interactively.The contents of groups.txt should be:1617The groups.txt file can be created with a plain text editor.Once you have this file,change the.txt file extension of distances.txt(linked above)to.pl and execute the script:perl distances.plLook at the contents of summary.dat to see the progression of COM distance between chain A and chain B over time.Make note of the configurations to be used for umbrella sampling,based on the desired spacing.That is,if you want0.2-nm spacing,you might find the following lines in summary.dat:500.600...1000.800You would then use conf50.gro and conf100.gro as the starting configurations of two adjacent umbrella sampling windows.Make note of all the configurations you wish to use before continuing.For the purposes of this tutorial,identifyingconfigurations with0.2-nm spacing will suffice,although in the original work a different(more detailed)spacing was used.Back:Energy Minimization and Equilibration Next:Umbrella SamplingG ROMACS TutorialStep Six:Umbrella Sampling SimulationsAfter having identified the initial configurations of the sampling windows,we can now conduct actual umbrella sampling simulations.We will need to generate a number of input files in order to conduct each of the necessary simulations. For example,if you have identified25configurations along the reaction coordinate,that means you will need25different input files for25independent simulations.You will simply have to call grompp to process this.mdp file for each of the conf.gro files you identified in the previous step.Many of the pulling parameters are the same as in the previous step,with the notable exception of pull_rate1,which has now been set to zero.We don't want to move the configuration along the reaction coordinate;instead we want to restrain it within a defined window of configurational space.Setting pull_start=yes means that the initial COM distance is the reference distance,and we do not have to define a reference (pull_init1)separately for each configuration.In this example,we will be sampling COM distances from0.5-5.0nm along the z-axis using roughly0.2-nm spacing.The following example commands may or may not be literally correct(the frame numbers may differ),but will serve as an example as to how to run grompp on separate coordinate files to generate all23 inputs(note as well that23is the amount of windows required to obtain0.2-nm spacing over roughly 4.5nm;in our original work,31asymmetric windows were used).You will also note that I have set gen_vel=no in the.mdp file.I have found that allowing the initial forces to govern the dynamics in each window is sufficient for a large,robust system such as this one.If this is not the case in systems with which you work,you will likely want to set gen_vel=yes and allow some time for equilibration in each sampling window.grompp-f md_umbrella.mdp-c conf0.gro-p topol.top-n index.ndx-o umbrella0.tpr ...grompp-f md_umbrella.mdp-c conf450.gro-p topol.top-n index.ndx-o umbrella22.tprNow,each input file should be passed to mdrun for the actual data collection simulation.Once all of the simulations are complete,you can proceed to data analysis.Back:Generating Configurations Next:Data AnalysisG ROMACS TutorialStep Seven:Data AnalysisThe most common analysis conducted for umbrella sampling simulations is the extraction of the potential of mean force(PMF),which will yield the∆Gforbindthe binding/unbinding process.The value ofﾎ濡is simply the difference between the highest and lowest values of the PMF curve,as long as the values of the PMF converge to a stable value at large COM distance.A common method for extracting PMF is the Weighted Histogram Analysis Method(WHAM),included in G ROMACS as the g_wham utility.The input to g_wham consists of two files,one that lists the names of the.tpr files of each window,and the other that lists the names of either the pullf.xvg or pullx.xvg files from each window.For example,a simpletpr-files.dat might consist of:umbrella0.tprumbrella1.tpr...umbrella22.tprAnd analogously for the list of pullf.xvg or pullx.xvg files,in eitherpullf-files.dat or pullx-files.dat.Note that the files must have unique names (i.e.,pullf0.xvg,pullf1.xvg,etc)or else g_wham will fail.We then run g_wham: g_wham-it tpr-files.dat-if pullf-files.dat-o-hist-unit kCalThe g_wham utility will then open each of the umbrella.tpr and pullf.xvg files sequentially and run the WHAM analysis on them.The-unit kCal option indicates that the output will be in kcal mol-1,but you can also get results in kJ mol-1or kT.Note that you may have to discard the first several hundred ps of the trajectory Bas equilibration(using g_wham-b),since we generated our starting configurations from a non-equilibrium simulation.Once the PMF converges,you should know how much time was required to equilibrate your system.You should end up with a profile.xvg file that looks like the following:Please note that the result you obtion may be different,since the spacing recommended in this tutorial is different from the spacing I actually used to generate this data in the original study.The overall shape of the curve should be similar,and the value of ∆G bind (calculated as the difference between theplateau region of the PMF curve and the energy minimum of the curve)should be close to -50.5kcal mol -1.The other output from the g_wham command will be a file called histo.xvg,which contains the histograms of the configurations within the umbrella sampling windows.These histograms will determine whether or not there is sufficient overlap between adjacent windows.For the types of simulations conducted as part of this tutorial,you may obtain something like the following:The above histogram shows reasonable overlap between windows from about 1.2-5nm of COM spacing;the overlap around 1nm (green and blue curves)indicates that more sampling windows are likely necessary to obtain good results from the WHAM algorithm.As it stands now,there is very little overlap between these two windows.Back:Umbrella Sampling Next:SummaryG ROMACS Tutorial SummaryYou have now hopefully been successful in conducting umbrella sampling simulations by generating a series of configurations along a reaction coordinate,running biasing simulations,and extracting the PMF.The .mdp files provided here serve as examples only,and should not be considered broadly applicable to all systems.Review the literature and the G ROMACS manual for adjustments to these files for efficiency and accuracy purposes.If you have suggestions for improving this tutorial,if you notice a mistake,or if anything else is unclear,please feel free to email me .Please note:this isnot an invitation to email me for G ROMACS problems.I do not advertise myself as a private tutor or personal help service.Happy simulating!--------------------------Md_pull.mdp---------------------------title=Umbrella pulling simulationdefine=-DPOSRES_B;Run parametersintegrator=mddt=0.002tinit=0nsteps=250000;500psnstcomm=1;Output parametersnstxout=5000;every10psnstvout=5000nstfout=500nstxtcout=500;every1psnstenergy=500;Bond parametersconstraint_algorithm=lincsconstraints=all-bondscontinuation=yes;continuing from NPT;Single-range cutoff schemenstlist=5ns_type=gridrlist= 1.4rcoulomb= 1.4rvdw= 1.4;PME electrostatics parameterscoulombtype=PMEfourierspacing=0.12fourier_nx=0fourier_ny=0fourier_nz=0pme_order=4ewald_rtol=1e-5optimize_fft=yes;Berendsen temperature coupling is on in two groupsTcoupl=Nose-Hoovertc_grps=Protein Non-Proteintau_t=0.10.1ref_t=310310;Pressure coupling is onPcoupl=Parrinello-Rahmanpcoupltype=isotropictau_p= 1.0compressibility= 4.5e-5ref_p= 1.0;Generate velocities is offgen_vel=no;Periodic boundary conditions are on in all directionspbc=xyz;Long-range dispersion correctionDispCorr=EnerPres;Pull codepull=umbrellapull_geometry=distancepull_dim=N N Ypull_start=yes;define initial COM distance>0pull_ngroups=1pull_group0=Chain_Bpull_group1=Chain_Apull_rate1=0.01;0.01nm per ps=10nm per nspull_k1=1000;kJ mol^-1nm^-2----------------------------------------------------------------------------------md-umbrella.mdp-------------------title=Umbrella pulling simulationdefine=-DPOSRES_B;Run parametersintegrator=mddt=0.002tinit=0nsteps=5000000;10nsnstcomm=1;Output parametersnstxout=50000;every100psnstvout=50000nstfout=5000nstxtcout=5000;every10psnstenergy=5000;Bond parametersconstraint_algorithm=lincsconstraints=all-bondscontinuation=yes;continuing from NPT;Single-range cutoff schemenstlist=5ns_type=gridrlist= 1.4rcoulomb= 1.4rvdw= 1.4;PME electrostatics parameterscoulombtype=PMEfourierspacing=0.12fourier_nx=0fourier_ny=0fourier_nz=0pme_order=4ewald_rtol=1e-5optimize_fft=yes;Berendsen temperature coupling is on in two groupsTcoupl=Nose-Hoovertc_grps=Protein Non-Proteintau_t=0.10.1ref_t=310310;Pressure coupling is onPcoupl=Parrinello-Rahmanpcoupltype=isotropictau_p= 1.0compressibility= 4.5e-5ref_p= 1.0;Generate velocities is offgen_vel=nogen_temp=310gen_seed=1;Periodic boundary conditions are on in all directionspbc=xyz;Long-range dispersion correctionDispCorr=EnerPres;Pull codepull=umbrellapull_geometry=distance;can't get PMF with directionpull_dim=N N Ypull_start=yespull_ngroups=1pull_group0=Chain_Bpull_group1=Chain_Apull_init1=0pull_rate1=0.0pull_k1=1000;kJ mol^-1nm^-2pull_nstxout=1000;every2pspull_nstfout=1000;every2ps--------------------------------------------------ions.mdp-------------;ions.mdp-used as input into grompp to generate ions.tpr;Parameters describing what to do,when to stop and what to saveintegrator=steep;Algorithm(steep=steepest descent minimization)emtol=1000.0;Stop minimization when the maximum force<1000.0kJ/mol/nm emstep=0.01;Energy step sizensteps=50000;Maximum number of(minimization)steps to perform;Parameters describing how to find the neighbors of each atom and how to calculate the interactionsnstlist=1;Frequency to update the neighbor list and long range forcesns_type=grid;Method to determine neighbor list(simple,grid)rlist= 1.4;Cut-off for making neighbor list(short range forces) coulombtype=PME;Treatment of long range electrostatic interactionsrcoulomb= 1.4;Short-range electrostatic cut-offrvdw= 1.4;Short-range Van der Waals cut-offpbc=xyz;Periodic Boundary Conditions(yes/no)------------------------------------------------------minim.mdp-------------;ions.mdp-used as input into grompp to generate ions.tpr;Parameters describing what to do,when to stop and what to saveintegrator=steep;Algorithm(steep=steepest descent minimization)emtol=1000.0;Stop minimization when the maximum force<1000.0kJ/mol/nm emstep=0.01;Energy step sizensteps=50000;Maximum number of(minimization)steps to perform;Parameters describing how to find the neighbors of each atom and how to calculate the interactionsnstlist=1;Frequency to update the neighbor list and long range forcesns_type=grid;Method to determine neighbor list(simple,grid)rlist= 1.4;Cut-off for making neighbor list(short range forces) coulombtype=PME;Treatment of long range electrostatic interactionsrcoulomb= 1.4;Short-range electrostatic cut-offrvdw= 1.4;Short-range Van der Waals cut-offpbc=xyz;Periodic Boundary Conditions(yes/no)-----------------------------------------------npt.mdp----------title=NPT Equilibrationdefine=-DPOSRES;position restrain the protein;Run parametersintegrator=md;leap-frog integratornsteps=50000;2*50000=100psdt=0.002;2fs;Output controlnstxout=1000;save coordinates every2psnstvout=1000;save velocities every2psnstenergy=1000;save energies every2psnstlog=1000;update log file every2ps;Bond parameterscontinuation=no;Initial simulationconstraint_algorithm=lincs;holonomic constraintsconstraints=all-bonds;all bonds(even heavy atom-H bonds)constrainedlincs_iter=1;accuracy of LINCSlincs_order=4;also related to accuracy;Neighborsearchingns_type=grid;search neighboring grid celsnstlist=5;10fsrlist= 1.4;short-range neighborlist cutoff(in nm)rcoulomb= 1.4;short-range electrostatic cutoff(in nm)rvdw= 1.4;short-range van der Waals cutoff(in nm);Electrostaticscoulombtype=PME;Particle Mesh Ewald for long-range electrostaticspme_order=4;cubic interpolationfourierspacing=0.16;grid spacing for FFT;Temperature coupling is ontcoupl=Berendsen;Weak coupling for initial equilibrationtc-grps=Protein Non-Protein;two coupling groups-more accuratetau_t=0.10.1;time constant,in psref_t=310310;reference temperature,one for each group,in K;Pressure coupling is onpcoupl=Berendsen;Pressure coupling on in NPT,also weak couplingpcoupltype=isotropic;uniform scaling of x-y-z box vectorstau_p= 2.0;time constant,in psref_p= 1.0 1.0;reference pressure(in bar)compressibility= 4.5e-5;isothermal compressibility,bar^-1;Periodic boundary conditionspbc=xyz;3-D PBC;Dispersion correctionDispCorr=EnerPres;account for cut-off vdW scheme;Velocity generationgen_vel=yes;Velocity generation is ongen_temp=310;temperature for velocity generationgen_seed=-1;random seed;COM motion removal;These options remove COM motion of the systemnstcomm=1comm-mode=Linearcomm-grps=System---------------------------------------------distance.pl-----------------#!/usr/bin/perl-wuse strict;#loop g_dist command-measure distance in each frame,write to a filefor(my$i=0;$i<=500;$i++){print"Processing configuration$i...\n";system("g_dist-s pull.tpr-f conf${i}.gro-n index.ndx-o dist${i}.xvg<groups.txt&>/dev/null");}#write output to single fileopen(OUT,">>summary_distances.dat");for(my$j=0;$j<=500;$j++){open(IN,"<dist${j}.xvg");my@array=<IN>;my$distance;foreach$_(@array){if($_=~/[#@]/){#do nothing,it's a comment or formatting line }else{my@line=split("",$_);$distance=$line[1];}}close(IN);print OUT"$j\t$distance\n";}close(OUT);#clean upprint"Cleaning up...\n";for(my$k=0;$k<=500;$k++){unlink"dist${k}.xvg";}exit;-----------------------------------------。