前言
计算单点突变自由能是分子稳定性改造的”老套路”。FoldX以及ddg_monomer application已经久经实战考验(具体用法参考前人总结:http://bioengx.hk1.91site.net/rosetta-ddg_monomer/)。今天我们就来说说Rosetta新一代的预测方法: Cartesian_ddG。
1 Cartesian_ddG方法概述
参考: Hahnbeom Park, Philip Bradley, Per Greisen Jr., Yuan Liu, Vikram Khipple Mulligan, David E Kim, David Baker, and Frank DiMaio (2016) “Simultaneous optimization of biomolecular energy function on features from small molecules and macromolecules”, JCTC.
Cartesian_ddG是一种新的采样方法,与ddg_monomer不同,对于骨架柔性的计算并不需要大量重复的强限制约束,而是采取了卡迪尔空间的优化来允许小幅度的骨架运动。
在计算∆∆G时,Cartesian_ddG方法使用两步Relax的方法:
-预先使用FastRelax进行优化,确定野生型以及点突变氨基酸最佳的侧链排布方式
-再使用卡迪尔空间版本的FastRelax对局部区域(点突变位置6埃范围内)进行彻底放松。
-评估野生型以及突变型蛋白的能量差,并使用能量函数特异性的校正因子对∆∆G值进一步矫正,以对应实验测定值单位kcal/mole。
效果:更快、更准。
Cartesian_ddG(使用opt-nov15函数)比ddg_monomer最佳Protocol(row16)要快上10倍有余。并且Cartesian_ddG预测∆∆G与实验∆∆G的皮尔森相关性从0.703升至0.743。
2 Cartesian_ddG的使用方法
Cartesian_ddG的使用方法十分简单。
-预优化野生型蛋白结构
-准备mutfile文件
-运行Cartesian_ddG
-分析∆∆G值
2.1 预优化野生型蛋白结构
创建relax_flag文件,文件内容如下:
-use_input_sc
-constrain_relax_to_start_coords
-ignore_unrecognized_res
-nstruct 20
-relax:coord_constrain_sidechains
-relax:cartesian-score:weights ref2015_cart # 务必设为ref2015_cart
-relax:min_type lbfgs_armijo_nonmonotone # 能量最小化的算法
-relax:script cart2.script
创建cart2.script文件,文件内容如下:(无需额外更改参数)
switch:cartesian
repeat 2
ramp_repack_min 0.02 0.01 1.0 50
ramp_repack_min 0.250 0.01 0.5 50
ramp_repack_min 0.550 0.01 0.0 100
ramp_repack_min 1 0.00001 0.0 200
accept_to_best
endrepeat
进行relax优化,打开终端并输入:
relax.mpi.macosclangrelease -s $pdb @relax_flag
打开relax的打分文件,查看最低能量的结构pdb编号,并以此作为出发的输入结构。
2.2 准备mutfile文件
单点突变的mutfile文件的格式如下:
注意!此处的89为pose序号,而非PDB中的残基号,设置错误一定会报错。
total 1 # mutfile中总突变数量
1 # 本轮突变氨基酸个数,单点突变设置为1.
T 89 A # 将89位的T突变为A
将文件保存为T89A.mutfile
2.3 运行Cartesian_ddG
Cartesian_ddG有两种运行模式: 蛋白稳定性预测模式(Protein stability mode)和 相互作用界面模式(Interface mode)。
1)蛋白稳定性预测模式(Protein stability mode)
创建cartddg_flag文件, 内容如下:
-ddg:iterations 5 # 默认为3,可以根据自身计算资源调整。
-ddg::cartesian
-ddg::dump_pdbs False # 是否输出突变后的蛋白PDB文件,推荐False,否则文件夹会很乱
-bbnbrs 1 # 骨架自由度,额外考虑线性邻居氨基酸的数目,1代表 邻近的3个氨基酸骨架自由度被考虑在计算中。
-fa_max_dis 9.0 # 控制范德华和溶剂化能量计算的范围,默认为6埃以内的氨基酸被考虑。
-score:weights ref2015_cart # 务必设为ref2015_cart
运行ddg计算,打开终端并输入,这么就会开始计算T89A的∆∆G。
cartesian_ddg.linuxgccrelease -s [inputpdb] @cartddg_flag -ddg:mut_file T89A.mutfile
2 )相互作用界面模式(Interface mode)
该模式开发者并未做详细测试,斟酌使用,此处笔者未做测试。如有需要请参考: https://www.rosettacommons.org/docs/latest/cartesian-ddG
3 结果分析
运行完毕后,文件夹中会产生T89A.ddg这个文件。打开后,文件内容如下:
COMPLEX: Round1: WT: -333.782 fa_atr: -840.140 fa_rep: ………….
COMPLEX: Round2: WT: -333.782 fa_atr: -840.140 fa_rep: ………….
COMPLEX: Round3: WT: -333.782 fa_atr: -840.140 fa_rep: ………….
COMPLEX: Round4: WT: -333.782 fa_atr: -840.140 fa_rep: ………….
COMPLEX: Round5: WT: -333.782 fa_atr: -840.140 fa_rep: ………….
COMPLEX: Round1: MUT_89ALA: -330.352 fa_atr: -834.516 ………….
COMPLEX: Round2: MUT_89ALA: -330.352 fa_atr: -834.516 ………….
COMPLEX: Round3: MUT_89ALA: -330.352 fa_atr: -834.516 ………….
COMPLEX: Round4: MUT_89ALA: -330.352 fa_atr: -834.516 ………….
COMPLEX: Round5: MUT_89ALA: -330.352 fa_atr: -834.516 ………….
此处Round代表第几次迭代,根据-ddg:iterations设置定义。
WT:代表本轮能量计算,野生型的能量为-333.782。
MUT_89ALA:代表本轮能量计算,突变型的能量为-330.352。
我们需要将WT和MUT_89ALA平均后,用MUT能量平均值减去WT能量平均值就可以得到T89A的∆∆G值,为3.43。
稳定性变化评判标准:(务必根据自身需要调整)
∆∆G < -1.0 代表点突变为蛋白带来稳定效应; ∆∆G > 1.0 代表点突变为蛋白带来不稳定效应;
备注: 如果需要批量分析和点突变扫描,请使用脚本生成多个mutfile,然后按照以上流程进行计算。
micronase 2.5mg uk – brand glucotrol where to buy dapagliflozin without a prescription
hzPJBYMtUZuc
order desloratadine 5mg – desloratadine for sale online buy albuterol generic
Hi,
I just visited bioengx.com and wondered if you’d ever thought about having an engaging video to explain what you do?
I can show you some previous videos we’ve done if you want me to send some over. Let me know if you’re interested in seeing samples of our previous work.
Regards,
Georgina
Unsubscribe: https://removeme.click/ev/unsubscribe.php?d=bioengx.com
purchase albuterol inhalator sale – order fluticasone online theo-24 Cr 400 mg drug
stromectol ivermectin – cefaclor pill where can i buy cefaclor
cheap azithromycin 250mg – buy tinidazole 500mg sale ciplox price
buy cheap clindamycin – order suprax 100mg buy chloromycetin without prescription
You actually make it appear really easy along with your presentation however I to find this topic to be actually
one thing that I feel I’d by no means understand.
It seems too complicated and extremely large for me. I’m looking forward for your subsequent post, I’ll try
to get the hold of it!
Feel free to surf to my web-site vpn coupon code 2024
Hi there! Would you mind if I share your blog with my zynga group?
There’s a lot of people that I think would really enjoy your content.
Please let me know. Thanks
Feel free to surf to my web site; vpn special code
amoxil us – purchase ceftin for sale order cipro 500mg generic
order augmentin 375mg without prescription – zyvox ca cipro price
atarax cost – generic amitriptyline 25mg endep ca
buy generic anafranil 50mg – order aripiprazole 20mg pills purchase sinequan sale
order clozaril pill – order frumil 5mg sale famotidine buy online
zidovudine medication – lamivudine online buy buy zyloprim sale
order metformin without prescription – order baycip lincomycin drug
lasix 100mg usa – atacand 16mg tablet captopril for sale
ampicillin online buy amoxil without a prescription buy amoxil for sale
flagyl ca – buy cefaclor 500mg zithromax for sale online
ivermectin 6 mg otc – sumycin us buy tetracycline 500mg without prescription
valacyclovir 1000mg drug – nemasole over the counter order acyclovir online cheap
ciprofloxacin 500 mg oral – buy doxycycline tablets order erythromycin 500mg pills
brand flagyl – order flagyl 200mg pills buy azithromycin 250mg sale
Hi there,
We run a Youtube, Instagram and Twitter/X service, where we can increase your subscribers/followers.
This is all done manually and so all subscribers and followers are real people.
Our prices start from just $60/month
If you are interested, please let us know, which of your social media profiles are you wishing to grow?
If you are not interested, no problem, just ignore and delete this email.
Kind Regards,
Sarah
baycip price – cephalexin buy online buy amoxiclav generic
buy cipro 1000mg generic – myambutol 600mg sale order augmentin 375mg for sale
lipitor 10mg canada buy atorvastatin 80mg pill order atorvastatin 20mg pill
VlrkbLCeda
创建relax_flag文件,文件内容如下:
-use_input_sc
-constrain_relax_to_start_coords
-ignore_unrecognized_res
-nstruct 20
-relax:coord_constrain_sidechains
-relax:cartesian-score:weights ref2015_cart # 务必设为ref2015_cart
-relax:min_type lbfgs_armijo_nonmonotone # 能量最小化的算法
-relax:script cart2.script
should be
-use_input_sc
-constrain_relax_to_start_coords
-ignore_unrecognized_res
-nstruct 20
-relax:coord_constrain_sidechains
-relax:cartesian
-score:weights ref2015_cart # 务必设为ref2015_cart
-relax:min_type lbfgs_armijo_nonmonotone # 能量最小化的算法
-relax:script cart2.script
Hi there,
We run a YouTube growth service, which increases your number of subscribers both safely and practically.
– We guarantee to gain you 700-1500 new subscribers per month.
– People subscribe because they are interested in your videos/channel, increasing video likes, comments and interaction.
– All actions are made manually by our team. We do not use any ‘bots’.
The price is just $60 (USD) per month, and we can start immediately.
If you’d like to see some of our previous work, let me know, and we can discuss it further.
Kind Regards,
Emily